Sq vol9 by Saltman Quarterly

Undergraduate Biological Research Journal UCSD Division of Bioloical Sciences

Volume 9 Nos. 1, 2 & 3 sq.ucsd.edu

YOU ARE WHAT YOU EAT The image depicts an H&E stain of intestinal epithelial cells, which line the track of the small intestine. This region of the gastrointestinal system is integral in the digestion and absorption of nutrients. Small projections into the intestinal lumen, called villi, aid in increasing the surface area available for absorption. These cells were isolated from mice a few hours after they were injected with Poly I:C, a synthetic analog for a dsRNA virus. The holes in the epithelial layer, and shortened villi length are indications of severe intestinal damage.

photo taken by Kim Vu

SALTMAN QUARTERLY Table of Contents 11

FEATURES 8 Fast Food and Obesity Iliana Nguyen

Anelah McGinness

New studies are linking physical exercise with brain development and stimulation. Could running make you smarter? 14 The Genetics behind Anorexia Lucy Luong

Long believed to be social disorders, current research is beginning to shed light on the genetic factors which drive the development of eating disorders.

Joseph Aleshaki

Seen as a light social activity, smoking hookah may post a greater threat to your health than previously believed.

How the fast food revolution established obesity as a primary health concern in America.

11 A New Reason to Exercise

Hooked on Hookah

Disease Modeling and Hope for Thousands Kevin Perez

Could induced pluripotent stem cells hold the answer to curing Rett Syndrome? 25

Axonal Regrowth Sharon Vongvanith

Model organisms are being used to study axonal regrowth, in hopes of treating neurological disorders.

RESEARCH 30 Is Algal Growth on Acropora

Coral Limited by Small Grazers? Jordan Arce

34 Effects of Habitat Complexity and

Fishing Pressure on Moray Eels (Muraenidae) Along Santa Elena Peninsula, Costa Rica Eric Garcia

39 The Exogenous and Endogenous

44 Spectrogram Correlation

Detector Optimization for the North Pacific Blue Whale (Balaenoptera musculus) B call Sarah Pfeil

48 Gender Affects Behavior,

Variation in Size, and Site Fidelity in Fireflies (Coleoptera: Lampyridae Photinus sp.) Kim N. Vu

Mechanisms Influencing the 53 Identification and Locomotive Activity and Burrowing Characterization of Circadian Behavior of Soldier Crab Mictyris Input Kinase (CikA) Localization longicarpus (Latreille, 1806) Partners in Syenchococcus on North Stradbroke Island, elongatus PCC 7942 Queensland, Australia Michelle Hoang and Stephen Leung Christopher J. Law

SENIOR HONORS THESES

REMEMBERING DR. PAUL SALTMAN

STAFF AND ACKNOWLEDGMENTS

LETTER FROM THE EDITOR

ne of UC San Diego’s most beloved professors, Dr. Paul Saltman emitted a charisma that stemmed from his passion for teaching. The torch that he lit upon arrival at our campus in 1967 continues to burn brightly, rooted in the world-class education he helped develop that students here receive. As our campus is still basking in the afterglow of its fiftieth anniversary festivities, the Saltman Quarterly program continues its ever-expanding celebration of the achievements of undergraduate

researchers in biology. Dr. Saltman firmly believed in the importance of undergraduate education outside of the classroom, pushing for undergraduates to perform research, but he understood that effective communication was equally important. The idea of uniting the two ideas led to the founding of Saltman Quarterly, which provided a forum for undergraduate researchers to publish their findings in the form of research manuscripts. As the Saltman Quarterly program expanded, new sections were added, initially through the inclusion of research abstracts written by undergraduates participating in the senior honors thesis program. More recently, students in our program have crafted articles in laymen’s terms to highlight research being presented at the Biological Sciences Student Research Showcase. These articles, featured in Under the Scope, are designed to spark the layman’s interest in biology research and the

endless possibilities discoveries can bring. Saltman Quarterly will always be forged in the spirit of Dr. Saltman, who inspired non-science majors with his Frontiers of Science courses. Perhaps the research showcased in the following pages will ignite your passion for field work to study the coral reefs of Heron Island or the fireflies of Costa Rica; the features articles might rekindle your interest in social and political issues that have a biological origin, such as the debate over the use of stem cells or the growing problem of obesity. I am proud to present to you Volume 9, imbued with a passion for biology passed down to us from a torch lit by Dr. Saltman.

Lawrence Ku Editor-in-Chief 2011-2012

Generously underwritten by Pictured on the cover: This photo of the Tecolote Ranunculus (Ranunculus asiaticus) was taken by Thomas LaCroix in the Flower Fields at Carlsbad Ranch. LaCroix is a senior at Eleanor Roosevelt College and is majoring in Literatures of the World.

The Saltman Family and supported by

The views expressed in this publication are solely those of Saltman Quarterly, its principal members and the authors of the content of this publication. While the publisher of this publication is a registered student organization at UC San Diego, the content, opinions, statements, and views expressed in this or any other publication published and/or distributed by Saltman Quarterly are not endorsed by and do not represent the views, opinions, policies, or positions of the ASUCSD, GSAUCSD, UC San Diego, the University of California and the Regents or their officers, employees, or agents. The publisher of this publication bears and assumes the full responsibility and liability for the content of this publication.

FEATURES

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a diverse collection of

FEATURES Hailed as one of the top research institutes in the country, UC San Diego is constantly expanding the field of biology. The Features section aims to highlight the ground-breaking work accomplished by undergraduates at UCSD.

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FAST FOOD &

OBESITY Iliana Nguyen

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staff writer

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background After smoking, obesity is the leading cause of mortality in the United States. This does not come as a surprise since obesity rates are higher than ever, with a staggering 33.8% of adults who are obese. Obesity statistics for children living in the U.S. are alarming as well, with approximately 17% (or 12.5 million) of children and adolescents ages 2 to 19 years characterized as obese. Obesity is determined through BMI (body mass index), a number that is calculated based on a person’s weight and height. Childhood obesity is a serious issue because children who are obese are predisposed to have many health problems and diseases in their childhood and adulthood. Although many factors can contribute to obesity, the underlying cause of excessive weight gain is an imbalance of energy. This occurs when more calories are taken in through diet than expended through energy-consuming activities. Kerri N. Boutelle, Ph.D., an Associate Professor of Pediatrics and Psychiatry at the University of California, San Diego, conducted a study to evaluate the nutritional quality of fast food purchased for children as well as the reasons for dining at the fast food restaurant. Dr. Boutelle, who studies childhood obesity and nutrition, explains why she chose to concentrate on youth, stating, “One out of every three children is either overweight or obese. We focus on children because the majority of overweight children will become overweight adults, and we have an opportunity to intervene with children earlier.” This is the first study published to evaluate the purchasing patterns of fast food for youth. Dr. Boutelle explains, “We were interested in studying fast food purchasing for families because it influences how children eat. We believe that educating parents may influence how they feed their children.” The number of meals eaten away from home is believed to contribute to extra calorie consumption. Overweight children and adolescents consume more foods away from home compared to other children. Due to its low nutritional value, cheap cost, and large portion size, fast food and consequent consumption habits has been considered one of the factors contributing to the obesity epidemic. Since its introduction to the United States, fast food has risen in popularity to become a prominent role-player in the everyday diet of children. According to studies, about 30% of children report consuming fast food on a typical day.

gender of the person for whom the item was purchased as well as the reasons for dining at the McDonald’s were inquired about. Answer choices included: convenience, cheap, adults liked the food, children liked the food, a reward for a hospital visit, hungry with no other options, kids wanted the toy, and that it is a usual place for lunch.

results To analyze the data, children were divided into three groups based on their ages: 2 to 5 years old, 6 to 11 years old, and 12 to 18 years old. Researchers then determined the nutritional content of the meals purchased for the different age groups as well as how the meals relate to daily nutrition requirements. On average, these meals contained a calorie count of 646 to 811 calories, which is 36% to 51% of the recommended daily calorie intake for youth. The children’s meals were found to be extremely high in sodium, with 866 to 1100 mg of sodium consumed per child. Most popular foods purchased for youth were French fries, soda, chicken nuggets, cheeseburgers, and hamburgers. A Happy Meal was purchased by a little more than half (53%) of the 490 youth. While McDonald’s does offer healthier options such as apple dippers, these choices were found to be less popular. The fruit and yogurt parfait, a heart-friendly option, demonstrated to be very unpopular, only purchased by 1.4% of the families. The top reasons for dining at McDonald’s were convenience and that the children enjoyed the food. About 50% of the families used fast food to reward their children for visiting the hospital. Surprisingly, 72% of adults reported that they liked eating the fast food. Even though this study was conducted at one fast food location, Dr. Boutelle states, “I don’t think the results would have changed in other locations. What may have changed is what kinds of families participated in the study. We had a wide range of families in this study, so I think it applies to a wide variety of families.”

“Studies have found that

obese children have a 70 to 80 percent chance of growing up to become obese adults.”

the study Over the course of six weeks, 544 families were surveyed during lunch time at the McDonald’s located in the Rady’s Children Hospital in San Diego, California. Researchers approached families with children who looked between the ages of 2 and 18 years. The families were asked to present their receipts and to complete a short survey after purchasing their food. Families were asked questions to explain their purchases: who each item was for, if items were shared, sizes ordered (small, medium, large), whether drinks were diet or regular, the type of combination meal purchased, if condiments were added, and lastly, if there were any changes made to their order. The age and sq.ucsd.edu

significance Childhood obesity is a life threatening condition because it significantly increases the chances of developing many health problems such as type 2 diabetes, stroke, heart attack, hypertension (high blood pressure), and various cancers. Childhood obesity may also lead to social, behavioral, and emotional problems. Obese children may suffer from low self-esteem, depression, and difficulty interacting with peers. Not only do children need to be aware of what they put in their mouths, but college students need to maintain healthy food habits as well. Dr. Boutelle explains, “College students are at an increased risk for weight gain because it is the first time they are on their own and they get to make all the decisions regarding their food intake. There are many cues in the college environment to overeat, and it is very VOL 9 • SALTMAN QUARTERLY

FEATURES Fun Facts 1. McDonald’s serves over 46 million people every day. This is more people than the population of Spain. 2. In order to burn off the calories after consuming a super sized coke, fries, and Big Mac, you would have to walk for 7 hours. 3. At Burger King, a 12 oz drink used to be a small. Now it is considered kid size. 4. Each day, 1 in 4 Americans visit a fast food restaurant. Source: Super Size Me (Film 2004)

important for college students to understand the impact of calories from fast food or other sources on their weight.” Many college students are no stranger to the dreaded “freshman fifteen” pounds gained during their first year of college from unhealthy eating habits. College students are free to eat whenever and whatever they want, even if this means devouring a tub of Ben & Jerry’s on a stressful day. Also, a majority of college campuses are located near or contain a fast food restaurant where students can buy food at a low cost. Many of these fast food chains are open during late hours, which is perfect for the student that has a midnight craving. Fast food is also ideal for the penny pinching college student who is always on the go—quick food at an affordable cost. However, college students need to be conscious of what they are putting in their mouths; what is best for their wallet is not necessary the best for their health, especially if it is a Big Mac which is high in sodium, high in calories, and has a low nutritional value. According to a study published in Pediatrics Journal, youth who consume fast food on a typical day consumed greater amounts of saturated fat, sodium, carbohydrate, and sugars and less dietary fiber than those who do not eat fast food. Consuming fast food also reduces the amount of healthy food options eaten. Children who consume fast food tend to consume more sugar beverages and less milk, as well as fewer fruits and vegetables containing starch. Fruits and non-starchy vegetables are an important part of the daily diet because they contain low energy density and high fiber content, which may protect against excess weight gain. Fiber also plays a major factor in regulating digestion as well as preventing heart disease and colon cancer. Consuming more than the recommended amount of sodium results in elevated blood pressure, which can increase the risk of many health problems. Blood pressure is the pressure of blood exerted against the walls of the arteries as the heart pumps blood. When the body ingests sodium, this raises osmolarity (the measure of the amount of solutes in a solution) because the concentration of sodium has increased while the volume of fluid in the body remains the same. Vasopressin is secreted, which causes the kidneys to absorb water and increases the blood volume. This causes an increase in blood pressure since the heart has to work harder to push the increased blood volume throughout the body. High blood pressure results in the heart exerting a greater pressure upon the arteries, causing them to stretch and expand to allow blood to flow easier. Overstretching creates weak spots in the artery walls, causing them to become more prone to ruptures, which may possibly lead to

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a stroke or an aneurysm. Also, congestive heart failure occurs when the heart muscle becomes worn out from working too hard to pump blood and, as a result, can no longer efﬁciently pump blood. Fast foods are high in cholesterol as well. Atherosclerosis occurs when the coronary arteries become harder and thicker from a buildup of plaque and cholesterol. This buildup causes blood ﬂow to become severely reduced or completely cut off. If portions of plaque disperse into the bloodstream, oxygen transport to the brain and heart can be disrupted, potentially resulting in a stroke or heart attack. Trans-fats escalate the risk of developing heart disease by increasing LDL cholesterol while lowering HDL cholesterol levels. LDL, known as “bad cholesterol,” is responsible for plaque build-up in arteries. HDL cholesterol, or “good cholesterol,” travels through the bloodstream and removes LDL. Eating large portions of energy-dense foods also play a factor in the obesity epidemic. Fast food restaurants are notorious for their “value meals,” which offers large portions at a low price. “Supersizing” is a widespread practice at many restaurants, which offers more food, such as a drink and a side dish, for a small additional cost. Recent studies have shown that greater portion size leads to greater intake and extra calories. When watching television or driving down the road, it is quite common to see some sort of advertisement or reference for a popular fast food chain. The fast food industry markets to children in hopes of fostering a fast food habit that will continue into adulthood. Therefore, it is important to realize the impact that fast food has on the daily diet of youth and its involvement in the obesity epidemic. However, does this mean that we should give up on eating at fast food joints entirely? Absolutely not. While hamburgers and chicken nuggets are tempting, some fast food restaurants do offer health conscious items such as the fruit and yogurt parfait or apple dippers. It is critical to keep in mind that moderation and variety are key for a balanced diet. According to the late Dr. Paul Saltman, “What makes food good or bad for you is volume, balance, and interaction. What you eat matters less than how much you eat, when you eat it, and what other foods you’re also eating.”

WRITTEN BY ILIANA NGUYEN. Iliana Nguyen is a

Biochemistry and Cell Biology major from John Muir College. She will graduate in 2013.

references

1. Boutelle, K., Email Correspondence, 1, Nov. 2011 2. Boutelle, PhD, Kerri N., Hanaah Fannin, BS, Ron S. Newfield, MD, and Lisa Harnack PhD. “Nutritional Quality of Lunch Meal Purchased for Children at a Fast-Food Restaurant.” Childhood Obesity 7.4 (2011): 316-21. Print. 3. Bowman, PhD, Shanthy A., Steven L. Gortmaker, PhD, Cara B. Ebbeling, PhD, Mark A. Pereira, PhD, and David S. Ludwig, MD, PhD. “Effects of Fast-Food Consumption on Energy Intake and Diet Quality Among Children in a National Household Survey.” Pediatrics 113.1 (2044): 112-18. Print. 4. Gifford Sawyer, Michael, Taylor Harchak, Melissa Wake, and John Lynch. “FourYear Prospective Study of BMI and Mental Health Problems in Young Children.” Pediatrics 128.4 (2011): 677-84. Print. 5. Rolls, PhD, Barbara J. “The Supersizing of America Portion Size and the Obesity Epidemic.” Nutrition Today 38.2 (2003): 42-53. Print. 6. Saltman, Paul, Joel Gurin, and Ira Mothner. The University of California San Diego Nutrition Book. Boston: Little, Brown, 1993. Print. 7. Schlosser, Eric. Fast Food Nation: the Dark Side of the All-American Meal. Boston: Houghton Mifflin, 2001. Print. 8. Silverthorn, Ph.D, Dee Unglaub. Human Physiology. 4th ed. San Francisco: Pearon Benjamin Cummings, 2009. Print. 9. Www.heart.org. Web. 20 Nov. 2011. <http://www.heart.org>.

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A New Reason to Exercise:

How Running Can Make You Smarter Anelah McGinness

staff writer

t should come as no surprise that exercise is good for your health; however, evidence from the past decade has shown that physical exercise may actually increase brain power as well. This link between fit bodies and fit minds has led to exciting implications about the potential of behavior to influence brain function.

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can running boost your GPA?

In June of 2010, the American College of Sport Medicine issued a press release on the results of a study involving 266 college students which showed that students who exercised more had higher GPAs. The study found that students with a GPA of 3.5 or higher were 4 times more likely to take part in vigorous physical exercise than students with a GPA lower than 3.0. Even after controlling for confounding variables such as gender and major, researchers found that students who exercised vigorously for 20 minutes, 7 days a week had GPAs that were, on average, 0.4 points higher than students who were sedentary – even if they studied for the same amount of time. Based on these results, the researchers were able to generate a formula to give a rough prediction of a student’s GPA based on variables including the amount of time he or she exercised. The study also suggested that students who were more physically active were also more dedicated to their studies. Students who studied more than 3 hours per day were 4 times more likely to engage in vigorous physical activity than students who studied 1 hour per day. And healthy young college students are not the only ones who stand to beneﬁt from research in this ﬁeld.

exercise and the aging brain At the University of Illinois, it was found that when a group of elderly people followed an exercise regimen of walking 3 times a week, MRI scans showed growth in many areas of the brain. Thus, a little exercise was able to produce visible improvements in these patients. And the more the better: those who had the most mental improvement were those with a higher VO2 max – an indication of physical ﬁtness in terms of how much oxygen patients consumed while exercising. So, older adults who want to keep their wits about them into their golden years would do well to take up some light jogging or aerobics, and it’s never too late to start. Even adults who had led sedentary lifestyles were able to expand their mental capacities and restore lost brain volume by engaging in moderate exercise. Many studies conducted among the elderly support these ﬁndings and have shown that adults who are physically active have better memories and are able to perform better on tests of mental function as they age.

more than just surveys

With the accumulation of evidence from surveys such as these, a causal explanation is rather appealing. After all, the results regarding college students seem to be in agreement with the common experience that students who are physically ﬁt tend to have lower stress levels and an improved sense of well-being in general that is highly conducive to academic success. However, correlation does not prove causation, and as encouraging as they are, the correlations between exercise and mental function can be easily subject to alternative explanations. For example, one cannot eliminate the possibility that students who exercise more may simply exhibit superior self-discipline and thus, are able to study more diligently. Then the results of the study from the American College of Sports Medicine could be construed to be a study showing a link between self-discipline and GPA. Therefore, exercise may indeed be recommendable for health reasons, but such surveys may not be convincing enough to persuade students to try boosting their grade point averages by hopping on the treadmill. However, a compelling body of evidence from the past several years has established a strong biochemical link between physical activity and the brain. In the lab of Dr. Fred Gage at the Salk Institute, studies led by Dr. Henriette Van Praag have found convincing evidence that physical activity can augment the birth of new brain cells and improve learning and memory in mice.

shiny new brain cells

It has only just begun to enter the realm of common knowledge that, contrary to what was previously thought, brain cells grow throughout adult life. In fact, new neurons are being born every day. This birth of newborn brain cells, termed “neurogenesis,” takes place in only two areas of the brain. One of those areas is the dentate gyrus – a particular group of brain cells in the hippocampus that plays a role in learning and memory. In order to distinguish new cells from old ones, mice were injected with a nucleic acid analog, BrdU, which could be incorporated into newly dividing cells and used to identify cells that were the products of adult neurogenesis. Cells that were newly born in the adult brains glowed when stained with a particular chemical that detects this analog. “What’s amazing about neurogenesis is that, in most parts of the brain, no new neurons are made after you are born. But in the hippocampus, more neurons are born almost every day,” says Chunmei Zhao, a member of the team in Dr. Gage’s lab studying this phenomenon. As one would expect, researchers in the Gage lab found that mice whose brains glowed with new cells due to higher rates of neurogenesis performed better on tests of cognitive function.

mazes and memory

Neurogenesis in the hippocampus of adult mouse. New neurons were labeled by retrovirus expressing green ﬂuorescent protein in 6 week old mouse. The mouse brain was taken 8 weeks after cell labeling, therefore green cells were about 7-8 weeks old. Young neurons can also be detected by immunohistochemistry using antibodies against immature neurons such as doublecortin. Photo courtesy of: www.school-clipart.com

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In the Gage lab, mice were engineered such that the injection of a chemical could inhibit the generation of new brain cells. These mice were then subjected to tests of learning and memory to afﬁrm the link between these new brain cells and brain function. In the Morris water maze test, the mice are put in a pool of milky water with some platforms just under the sq.ucsd.edu

FEATURES surface so the mice cannot see them. As it turns out, mice do not like water; in order to get out, they have swim around to find out where the platform is. When they are put back in the same pool later, mice show they have learned by finding the platform more quickly. Mice in which neurogenesis had been inhibited were less able to learn where the platform was, spending more time swimming around randomly even though they had been placed in the exact same situation a few times before. In addition to the water maze test, mice in this experiment were subjected to a test of mental function called “contextual fear extinction.” In this test, mice are put in a particular environment with certain markers. Then they are given a shock so that they will learn to associate that environment with fear, causing them to freeze when placed there. Mice with poorer memories do not learn to exhibit this freezing behavior as quickly as mice with better memories – that is, they are worse at remembering what happened to them the last time they were in a given environment. This is exactly what happened with the inhibited mice. Unable to grow new brain cells, mice showed less fear of places they should associate with pain. The findings of these tests have been reproduced in other labs, serving as strong evidence to support a role for neurogenesis in learning and memory.

running and neurogenesis

In 1999, the Gage lab found that, compared to mice that were sedentary, mice that exercised voluntarily by running on a wheel had at least twice as many surviving newborn neurons as did mice who did not have the option of exercising. The area of their brains responsible for learning and memory was growing in response to the aerobic exercise. In order to reinforce the connection between exercise, neurogenesis, and the “intelligence” of a mouse, the mice in the study also took the Morris water maze test. Mice that were in the running group signiﬁcantly outperformed their peers – even mice that were supposed to exercise by swimming. Thus, the same group of mice that showed a higher rate of newborn brain cell growth also showed improved ability to learn and remember. “And the best thing is, it is never too late to exercise,” says Dr. Zhao, “even in old mice you can still see a very pronounced effect.” The size of the hippocampus and the rates of neurogenesis decline with aging; however, these dimensions can actually be rescued by voluntary running. Another experiment led by Dr. Van Praag in 2005 found that, in aged mice which had led sedentary lifestyles and exhibited this decline, neurogenesis could be restored to 50% of the levels that young exercising mice showed. Thus, exercise not only improves neurogenesis over the course of a lifetime but is capable of rescuing neurons from decline – partially restoring the minds of aged mice to a younger, ﬁtter time in the timeline of their mouse lives.

but does this work for humans?

Since these landmark studies in mice, more work has been done to find evidence that exercise increases neurogenesis and brain function in living humans. In 2007, a collaboration between the Gage lab and the lab of Dr. Scott Small of Columbia University found that, in a group of people aged 21 to 45 who exercised for 12 weeks, subjects who exercised performed better on a variety of tests of mental agility. MRI scans of the brains of these same patients showed that blood flow increased in the dentate gyrus specifically – the very same region where new brain cells are born. Furthermore, autopsies sq.ucsd.edu

of patients showed that there were higher rates of neurogenesis in patients who increased blood ﬂow to this region through exercise. Thus, researchers have found evidence that the connection between exercise, neurogenesis, and brain power may apply to a living, breathing human being just as it applies to mice. “Adult neurogenesis was initially thought of as way in which the brain may be able to repair itself after disease or injury,” says Dr. van Praag, now with the National Institute on Aging. “There is now both correlative and causal evidence [that] exercise enhances neurogenesis and is correlated with improved performance on both general hippocampus dependent tasks (maze learning) and tests tailored more speciﬁcally to test the function of the dentate gyrus (spatial pattern separation learning).”

moving forward

Researchers continue to conduct more experiments in order to refine the biological connection between physical and mental fitness. Scientists still do not know for sure what exactly it is about running that can cause newborn brain cells to grow in the adult hippocampus. It is not known whether the effects are simply due to increased rate of blood flow, the growth of new blood vessels, or the generation of molecules during exercise that have a particular effect on the adult brain. “All those factors may play a role. We are looking for the trigger in the periphery (muscle),” says Dr. van Praag, referring to her recent research in which the injection of endurance factors enhanced cognition and elevated adult neurogenesis in the dentate gyrus of mice. All in all, the evidence that we have so far is rather encouraging: it does indeed seem that exercise is good for your brain. “For the general population, the research may give another reason to exercise,” adds Dr. Van Praag, “I exercise more myself, though I was always rather into sports.” So for students, exercise may be as much a part of preparing for that midterm as working on problem sets; for older adults who want to keep their minds from slowing down, going for a run may be as helpful as buying a fancy memory game. In the end, the phenomenon of neurogenesis brings us back to the core idea that our behavior, what we do on a daily basis, can have a tangible, biological effect on the development of our minds. Now that’s something to think about.

WRITTEN BY ANELAH MCGINNESS. Anelah McGinness is a Physiology and Neuroscience major, pursuing minors in Spanish Literature and Health Care/Social issues from Revelle College. She will graduate in 2013.

references 1. Parker-Pope, T. (2010, June 3). Vigorous exercise linked with better grades. New York Times. 2. Hertzog, C., Kramer, A. F., Wilson, R. S., Lindenberger, U. (2009). Fit body, fit mind. Scientific American, 20(4), 24-31. 3. C. Zhao, personal communication, 2011. 4. Deng, W., Saxe, M.D., Gallina, I. S., Gage, F. H. (2009, October 28). Adult-born hippocampal dentate granule cells undergoing maturation modulate learning and memory in the brain. J Neurosci, 29(43), 13532-42. 5. Van Praag, H., Christie, B. R., Sejnowski, T. J., Gage, F. H. (1999, November 9). Running enhances neurogenesis, learning, and long-term potentiation in mice. Proc Natl Acad Sci U S A, 96(23), 13427-31. 6. Van Praag, H., Kempermann, G., Gage, F. H. (1999, March). Running increases cell proliferation and neurogenesis in the adult mouse dentate gyrus. Nat Neurosci, 2(3), 266-70. 7. H. Van Praag, personal communication, 2011.

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The Genetics Behind Anorexia

The search for an eating disorder treatment, right down to a single gene. Can manipulation of DNA lead to solutions for anorexia? Lucy Luong

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rom television screens, magazines, billboards, movies, and more, the image of a slim figured, flawless skinned, curvaceous female body is present. The picture of the “perfect woman” is seen by the masses everywhere in the media today. Many women who view these images see them as a message that promotes achieving the seemingly flawless body. However, the ideal figure that many women attempt to attain is nothing more than a highly edited image altered to look faultless and attractive to gain audience attention. Nonetheless, the message still spreads rapidly to women everywhere as they work to develop a slim figure. The self-consciousness that results from comparing their own bodies to the highly edited ones can lead to various unhealthy physical and mental activities, such as over-exercising, extreme dieting, binge eating, starvation, depression, and high anxiety. These actions and concerns about body imperfections prove to be the initial characteristics of individuals suffering from anorexia nervosa and bulimia nervosa. Anorexia nervosa and bulimia nervosa are two very serious types of eating disorders. An eating disorder is a complex, psychiatric problem involving irregular eating habits and extreme concern over body weight. People with eating disorders undergo more than just physical problems; they face behaviors that cause them to develop negative attitudes towards their bodies’ appearance and weight. This attitude and behavior are certain symptoms of anorexia and bulimia. While both are eating disorders, anorexia nervosa and bulimia nervosa have one major difference. Individuals with anorexia tend to refrain from eating while people with bulimia go through a pattern of binge eating or overuse of laxatives in order to lose weight.

overview of the study

Bloss explains that the study ﬁrst began from the stirring questions of why only a small percentage of women spontaneously recover from their eating disorders. Anorexia nervosa and bulimia nervosa are very complicated disorders to treat, and it was hypothesized that a person’s genetic makeup could play a part in answering the question of why a small number of people overcome their eating disorders

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Anorexia and bulimia are mental health illnesses that have a significantly high mortality rate. The National Association of Anorexia Nervosa and Associated Disorders gathered mortality statistics on these eating disorders. According to the association, 5 to 10% of eating disorder patients die within ten years, and 18 to 20% die twenty years after identified with the disorder. About 30 to 40% of patients with serious eating disorders are able to have a full recovery. The 2009 American Journal of Psychiatry estimated mortality rates were 4.0% for anorexia nervosa, 3.9% for bulimia nervosa, and 5.2% for other uncategorized eating disorders. The preceding statistics show the evident difficulty of recovering from eating disorders. Among those who develop an eating disorder, a large proportion remains ill, even if only mentally, throughout life. Recent research done by scientists at University of California, San Diego, School of Medicine and Scripps Translation Science Institute (STSI) has pointed out that genetic variations may be playing a role in influencing one’s recovery from an eating disorder. Leading the project are Cinnamon Bloss, Ph.D., STSI assistant professor and first author of the study’s paper, Walter H. Kaye, M.D., UCSD’s Eating Disorder Treatment Program director and psychiatry professor, and Nicholas J. Schork, Ph.D., bioinformatics and biostatistics director at STSI and professor at The Scripps Research Institute. From their studies, these researchers discovered certain genetic variations that cause difficulty for individuals to recover from anorexia nervosa or bulimia nervosa. With this new knowledge, further research can be used to create new and effective treatments specific to a patient’s genetic composition and ultimately to treat patients with anorexia nervosa and bulimia nervosa. while many others have a harder time recovering. “Anorexia and bulimia likely stem from many different causes such as culture, family, life changes, and personality traits,” said Bloss. “But we know biology and genetics are highly relevant in terms of cause and can also play a role in how people respond to treatment. Understanding the genetics behind these conditions is important, be sq.ucsd.edu

FEATURES because it could eventually help us tailor treatment based on the person’s genetic makeup, with the goal of more personalized and effective treatments.” The study conducted is the Price Foundation Candidate Gene Project. This project assesses speciﬁc genes in a candidate that may play a role in the genetic inability to recover from an eating disorder. The Price Foundation Collaborative Genetic Dataset provided the project with 1878 women to participate in the research study; a total of 1201 women were diagnosed with an eating disorder and 677 women were used as controls. Researchers at UCSD and STSI were in charge of designing the study. The study consisted of two cohort groups, one group of women over 25 years old and another group of women under 25 years old. The women from each group lived in roughly the same period of time; thus the study made sure outlying factors were taken into consideration. Both generations consisted of women who either have eating disorders or have recovered from one, allowing for the comparison and location of genes involved in preventing women from recovering. With blood samples taken from the 1878 women studied, the researchers were able to extract DNA samples and measure the genetic variants in the DNAs. Approximately 350 genes were observed, and a total of 5151 single-nucleotide polymorphisms (SNPs) within these genes were analyzed. SNPs are DNA sequences that vary in one single nucleotide (A, T, G, or C) in the sequence. For example, DNA sequences GCAT and GTAT vary in only one single letter; the second single nucleotide is either C or T. These differences result in SNPs. Along with the genetic makeup of the individual women, observable traits, also known as phenotypes, were examined as well. Hereditary traits such as high anxiety, which can lead to perfectionism, associated with eating disorders were taken into consideration in the research to analyze the role of genetics in these women’s recovery process.

the role of GABA genes The scientists from UCSD and STSI discovered 25 statistically significant SNPs that play a role in the women’s eating disorder recoveries. Ten of the twenty-five SNPs were haplotypes, a mix of alleles inherited together, and located close to each other on a chromosome. They concerned SNPs of GABA genes. GABA stands for gamma-aminobutyric acid, which is a chemical that controls the central nervous system. A type of neurotransmitter, GABA genes are responsible for many psychological functions and functions regarding the brain. They play a role as a nerve transmitter sending messages to the brain. One of these messages they are responsible for happens to be anxiety or worry, one of the important characteristics that causes eating disorders in individuals. From the study, GABA genes have shown some amount of responsibility for the outcome of women’s recovery from anorexia, bulimia, or other eating disorders. According to the study, women who did not recover from long-term eating disorders displayed higher frequencies of genetic variation in their GABA genes. GABA genes have multiple variations, and various versions can either be detrimental or protective to an individual. Whether or not these genes will benefit an individual simply depends on heredity and chance. Those who are lucky will receive the protective variation of the GABA gene from their parents that causes them to have less anxiety. However, those who are unlucky will inherit the detrimental variation of the GABA sq.ucsd.edu

gene that causes them to be more anxious. Bloss stated, “In terms of our ﬁndings, individuals with eating disorders who inherited the unlucky variant were more likely to remain ill with an eating disorder for a longer period of time relative to those with the lucky variant, who were more likely to recover quickly (i.e. before age 25). ” Although not completely proven, these genes play a responsible role in individual behavior disorders and not just in brain functions. Findings from other research have shown that GABA genes are related to anxiety levels. In this particular study by UCSD and STSI, women with eating disorders who displayed poor recoveries from eating disorders were the most anxious out of all the women in the cohort study. This is because a beneﬁcial variation of the GABA gene is more common in people who recover from their eating disorder. The research study showed that the control women had distinct SNPs that varied from the women who could not recover from their eating disorders. This distinction substantially supports the hypothesis that genetics does have a crucial role in women’s recovery from anorexia, bulimia, and other eating disorders. In addition, there is an intronic SNP located on chromosome four of the GABRGI gene. This intronic SNP does not produce any proteins and rather strongly connects to chronic eating disorder symptoms. Studies show that there may be genes that will incline an individual to undergo a chronic eating disorder but further research must be done still.

a call to new treatment

Through these ﬁndings, researchers now have greater insight towards the role of genetics in eating disorders. There are multiple genes that cause eating disorders. Substantial evidence shows that people who develop eating disorder tend to have certain inherited personality trait such as anxiety, from family members who share a common history. Genes, therefore, do have an inﬂuence on a person’s susceptibility to recover from an eating disorder. The slightest GABA gene variation can cause a person to inherit greater anxiety and become more vulnerable to eating disorder. With the support of future research, different types of drugs will be developed that target the GABA genes. As the information about the role of genetics in eating disorder recovery becomes more recognizable, treatments of eating disorder will be developed for these individuals’ needs.

WRITTEN BY LUCY LUONG. Lucy Luong is a senior

at The Preuss School UCSD. She will graduate from high school in 2012 and attend Dartmouth College next year.

references 1. C. Bloss, personal communication, December 3, 2011. 2. Bloss, C. (2011). Genetic Association of Recovery from Eating Disorders: The Role of GABA Receptor SNPs. Neuropsychopharmacology. 3. Crain, C. (2011, August 15). Team of International Scientists Finds Genetic Pattern Associated with Poor Recovery from Eating Disorders. News & Views Online weekly of The Scripps Research Institute. Retrieved from http://www.scripps.edu/ newsandviews/e_20110815/schork.html 4. Eating disorders Statistics. (2011, December 4). National Association of Anorexia Nervosa and Associated Disorders, Inc. Retrieved from http://www.anad.org/getinformation/about-eating-disorders/eating-disorders-statistics 5. Kain, D. (2011, July 26). New Study Sheds Light on Role of Genetics in Recovering from Eating Disorders. UC San Diego News Center. Retrieved from http://ucsdnews.ucsd.edu/newsrel/health/20110726EatingDisorders.asp

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FEATURES Joseph Aleshaki

staff writer

alking into a hookah lounge, you immediately experience sensory overload: your eyes narrow to focus through air that is marbled with hazy smoke, your ears are bombarded with laughter from patrons’ conversations, and your nose is filled with a myriad of exotic fruity smells. A sense of excitement permeates the electric atmosphere of chatter, accompanied by a feeling of relaxation amidst the comfort of friends. Tall hookahs with flashy, intricate designs stand prominently at every table as smokers breathe intoxicating flavors of tobacco. Unbeknownst to you, the history of the hookah dates back nearly 2000 years. The modern-day design of the hookah is credited to 17th century Turks living under the reign of Murat IV. It was in Turkey where hookah design was revolutionized by the additions of metal and glass to increase structural integrity. A hose attached to the base of the hookah was also added, allowing users to inhale vapors more easily. Besides the hose, the other main parts of the hookah are the bowl, used to hold the flavored tobacco; the stem, a long pipe that carries smoke from the burnt tobacco; and the base, which holds the water used to cool and “filter” the smoke passing through the hose. When a user inhales smoke from the hose, air is pulled under the coals, increasing their temperature. This increase in temperature burns the flavored tobacco, causing smoke to diffuse through the stem. The smoke is eventually vacuumed through the hose to the user. Surprisingly, hookah’s social attraction has not changed since ancient times, as evinced from its surge in popularity today. Despite unfavorable social norms surrounding smoking due to the negative effects of tobacco use, people aren’t holding back from the habit. “Smoking hookah feels much cleaner than cigarettes,” says Omar, a native Egyptian student at the University of California, San Diego. Omar, as with many students across the United States and beyond, has bought into misconceptions of hookah smoking as a “cleaner” and “safer” alternative to cigarettes — a misconception that has fortunately been debunked.

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mistaken perceptions of hookah use Professor Wael Al-Delaimy, chief of the Division of Global Health in the UCSD School of Medicine’s Department of Family and Preventative Medicine, has been at the forefront of tobacco research since 1990. “Tobacco use has been my career because it is the single most preventable cause of disease,” he says, “so stopping it would have extremely beneﬁcial outcomes to the health of our society.” He too understands that one of the main culprits contributing to hookah’s meteoric rise is the belief students such as Omar hold: hookah smoking is less harmful because smoke is ﬁltered by the water contained in the base. However, passing bubbles through water does not change its content. According to Al-Delaimy, as air bubbles pass through the water, its contents of tobacco carcinogens are still maintained. Unfortunately, this misperception of hookah as a cleaner form of tobacco smoking has caused people to increase the length of time they spend smoking. When compared to cigarette smoking, the number and volume of puffs from using hookahs are about ten times higher, according to a study conducted by Professor Alan Shihadeh from the American University of Beirut. To make matters worse, hookah smoke also contains 36 times the amount of nicotine and even higher concentrations of heavy metals such as cadmium and lead. Since the average smoking session lasts around 47.5 minutes, much more smoke is expelled into the air compared to a cigarette smoking session. Thus, second-hand smoke poses a great danger to non-smokers in all situations, who tolerate the smoke for the company of friends.

determinants of hookah use Friends exert the greatest influence on high school hookah use, according to a landmark study Al-Delaimy conducted in conjunction with San Diego State University. It was the first of its kind to address patterns of initiation, use, and cessation in a population of hookah users. More importantly, it is the first survey of high school students to compare demographics, risk perception, and other behavioral factors between hookah users and non-users. Interestingly enough, Al-Delaimy discovered that 50.3% of the high school students surveyed first learned about hookah from friends. Of those students who had heard of hookah, 59.5% believed it more socially acceptable, and 46.3% believed it safer than cigarettes. In addition, a correlation was found between frequent hookah users and hookah accessibility: 22.3% of frequent hookah users reported that they first learned of hookah by seeing a hookah lounge. Al-Delaimy believes that the proximity of hookah lounges to high schools is increasing access to the behavior. In addition, 13.5% of all minors surveyed said they had visited a hookah lounge. Illegal visitation of hookah lounges and use of tobacco by minors may suggest this is a widespread phenomenon that endangers the health of adolescents and young adults.

characteristics of current hookah users Al-Delaimy also began establishing demographic statistics of current hookah users in a comprehensive paper published in Tobacco Induced Diseases, which surveyed all hookah-serving venues in downtown San Diego, California. Al-Delaimy conducted the survey

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using brief questionnaires which consisted of questions about each participant’s age, gender, and ethnicity. To assess the association between cigarette and hookah smoking, participants were asked about cigarette smoking, how often they smoked a day, and the brand of cigarettes they used. Hookah users were asked about their favorite tobacco ﬂavor and frequency of their hookah use. To assess the perceptions of hookah use, they were asked for their opinion about the harmfulness of hookah smoking compared to cigarette smoking. Much to Al-Delaimy’s amazement, 58.3% of hookah users surveyed believed smoking hookah was less harmful than smoking cigarettes.

nicotine addiction Although, as mentioned earlier, the effects of smoking hookah are considerably worse than those of smoking cigarettes, the mechanism for addiction is the same. Nicotine from the smoked tobacco in the base of the hookah binds to nicotinic acetylcholine receptors (nAChRs) of neurons in a speciﬁc part of the midbrain called the ventral tegmental area (VTA). This binding subsequently activates VTA neurons to release dopamine, a hormone which positively regulates reward centers of the brain and gives hookah smokers the feeling of pleasure associated with smoking. nAChRs have important functional properties that contribute to their physiological effects, including activation, desensitization, and upregulation following nicotine exposure. There are three main neurons in the VTA that express nAChRs and induce these physiological effects: dopamine neurons, GABA neurons, and glutaminergic presynaptic terminals. Most important to the regulation of nicotine exposure are the GABA neurons, which are inhibitory neurotransmitters that exercise inhibition of VTA neurons. However, nicotine is an inhibitor of GABA neurons that cause smoking addictions to arise because other VTA neurons will continue to release dopamine with little regulation. Thus, GABA neuron desensitization by nicotine causes hookah smokers to continue smoking until they achieve a desirable level of pleasure, eventually limiting their own ability to produce dopamine. This causes hookah and tobacco smokers to become increasingly reliant on their smoking habits to activate their reward centers and “feel good.”

prevention Al-Delaimy is in disbelief at the speed at which hookah use has risen, making him a steadfast advocate for hookah prevention: “Not many Americans were doing it 10 years ago. Now, it’s picking up interest amongst people who haven’t even smoked!” Because hookah is a relatively new phenomenon, hookah prevention and education are still in their early stages. According to California law codes, currently any establishment posing as a tobacco shop may allow customers to smoke their purchases on-site legally. Hookah lounges have proliferated for precisely this reason. “What hookah lounges have done is use a loophole in the law that says that selling tobacco in a tobacco shop allows customers to smoke their purchase right there after buying it. This has given the impression that it’s ok to smoke it because the lounges are right there,” says Al-Delaimy. He believes the best solution to reversing the trend in hookah use is to close this loophole. “Once we ban smoking even in places that sell it, that would close the loophole and make it less appealing for people to go out and smoke. It would send the message that hookah is not allowed because it is dangerous. We need legislation passed sooner sq.ucsd.edu

FEATURES Anatomy of a Hookah Bowl Holds tobacco and charcoal burned on top during smoking; made of clay, marble

1 Plate Ash tray, used for “dead” coals from previous smoking sessions

Body, gasket, valve Body is a hollow tube with gasket at bottom; gasket has opening for hose and seals connection of body with water jar

Water jar Smoke from tobacco passes through jar, gaining moisture and lowering its temperature before it reaches hose

Hose Slender tube that allows smoke to be drawn; its end is typically fitted with a designed, metal wooden or plastic mouthpiece

How it Works...

Inhaling via the nose, air is pulled through coal and into bowl; hot air from charcoal burns from tobacco, producing smoke

Smoke passes through body tube, extending into water jar; it bubbles up to the top of the water jar and into a channel connected to the hose

rather than later.” In the meantime, Al-Delaimy is continuing to work towards the formation of a tobacco-free society. “My ultimate goal is to stop this trend and make it be something of the past…to have a day where there is no tobacco use, eliminating all preventable diseases associated with [tobacco].”

WRITTEN BY JOSEPH ALESHAKI. Joseph Aleshaki is a Human Biology major from Thurgood Marshall College. He will graduate in 2013.

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When a smoker inhales from the hose, pressure in the jar changes pulling more air through the charcoal and continuing the process

references 1. Aljarrah, K., Zaid Q.A., Al-Delaimy, W.K. (2009). Perceptions of Hookah Smoking Harmfulness: Predictors and Characteristics among Current Hookah Users. Tobacco Induced Diseases, 5(1), 16. 2. Hookah History. (2012, January 17). Hookah History. Retrieved from http://www. hookahcompany.com/hookah_history.htm 3. Mansvelder, H.D., McGehee, D. (2002). Cellular and Synaptic Mechanisms of Nicotine Addiction. Journal of Neurobiology, 53(4), 606-17. 4. Messer, K., Trinidad, D.R., Al-Delaimy, W.K., Pierce, J.P. (2008). Smoking Cessation Rates in the United States: A Comparison of Young Adult and Older Smokers. American Journal of Public Health, 98(2), 317-22. 5. Smith, J.R., Edland, E.D., Novotny, T.E., Hofstetter, C.R., White, M.M, Lindsay, S.P., Al-Delaimy, W.K. (2011). Increasing Hookah Use in California. American Journal of Public Health, 101(10), 1876-879. 6. Smith, J.R., Novotny, T.E., Edland, S.D., Hofstetter, C.R., Lindsay, S.P., Al-Delaimy, W.K. (2011). Determinants of Hookah Use among High School Students. Nicotine Tob Res, 13(7), 565-572. 7. Adolescents’ perceptions about quitting and nicotine replacement therapy: ﬁndings from the California Tobacco Survey. Retrieved from http://www.ncbi.nlm.nih.gov/ pubmed/16549315

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DisEAsE MODELiNG AND HOPE FOR THOUSANDS

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o say that we are in peacetime is a severe misconception, since we are always at war. Not with other countries or even with members of our own species but with our very own bodies. The fight against microscopic antagonists is never ending, and it may even seem like a losing battle, especially when there are enzymes in our own body causing potential conflict. Fortunately, we have the power of human innovation on our side. Whenever a disease erupts, researchers try to meet it with scientific exploration and application. Even in the field of mental diseases, an area of relatively unknown medical expertise, scientists have made several significant discoveries. Amongst these diseases is Rett syndrome, an ailment categorized as an autism spectrum disorder (ASD). With the creation of induced pluripotent stem cells (iPSCs), our potential ability to combat Rett syndrome looks very promising.

Kevin Perez

staff writer

causes and symptoms Rett syndrome is a developmental disorder of an autistic nature that stunts the patient’s social capacity, preventing them from properly exhibiting normal social behaviors. It is understood that Rett syndrome originates from a genetic mutation in the MECP2 gene, located on the X chromosome. Sporadic mutations, mutations that occur in the child independent of parental inheritance, account for over 95% of these cases. Often times Rett syndrome is diagnosed based on clinical observation, similar to how a psychiatrist might diagnose a patient for ADHD if they are unable to focus on one object for a certain length of time. These observations are based on the common symptoms of the syndrome. In addition to seizures, gastrointestinal disorders, and developmental discrepancies, patients with Rett syndrome often display no abnormal verbal and social behavior, which explains why it is considered to be an ASD. Given that the syndrome is caused by mutations during development, potential for preventing or treating such a disease might not seem feasible. The most threatening aspect of the syndrome is the spontaneity in its sq.ucsd.edu

development, which is mostly caused by sporadic mutation. Luckily, researchers at the University of California San Diego observed the potential in the application of induced pluripotent stem cells (iPSCs) and began using these techniques to uncover the heart of the mutation at the MECP2 gene.

induced pluripotent stem cells Assistant Professor of Pediatrics, Alysson R. Muotri, Ph.D., led the research that was conducted at the UCSD School of Medicine. After his research, he concluded that induced pluripotent stem cells would allow scientists to observe how Rett syndrome develops at the molecular level. The synthesis of iPSCs is a process aimed at reprogramming cells to express genes that researchers wish them to express. Scientists take somatic cells, the most common of which would be a regular skin cell, inject them with retroviral reprogramming vectors, and express genes that would code for a pluripotent state. The products of the experiment are cells which behave identically to embryonic stem cells. What makes iPSCs so remarkable is the resource of potential cells that it can produce. One could create neurons from stem cells and then view the mutation of MECP2, in essence, witnessing the development of Rett syndrome at the molecular level. This is exactly what Muotri and his team did. VOL 9 • SALTMAN QUARTERLY

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the research and disease modeling

RTT cells did not have any signiﬁcantly decreased survival rates than those of the control cells, which meant that they formed at the same rate as normal neurons. Next, the researchers looked at the MeCP2 gene and what occurred to it during differentiation. They found exactly what they had predicted, that in the RTT neurons, a mutation occurred in the MeCP2 gene which would not produce full length MeCP2 proteins. Muotri’s team had successfully modeled how and when Rett syndrome would manifest in a patient at the molecular level. By isolating the moment of mutation, they were able to begin work on the reversal of this process. Muotri and his team began suppressing the nonsense mutations in the genome in hopes that it would increase levels of MeCP2 expression. They treated the RTT cells with gentamicin, an antibiotic which would bind to a speciﬁc area of rRNA and produce full length proteins. To their amazement, they found that MeCP2 protein levels increased after one week, showing great potential for future progress.

Brazilian born Alysson Muotri came to the United States in search for a lab research position. While his background was in genetics, he always had a strong passion for neuroscience and the development of the human brain. After learning about the invention of induced pluripotent stem cells and what Professor Yamanaka and his team had been able to do at Kyoto University, Muotri knew immediately that he wanted to apply the idea of stem cells to neuroscience. Eventually, this passion brought him to the La Jolla coast, where his research promptly began. He began by examining the molecular fate of the neuron, beginning at the stem cell phase and following through its development into a functioning adult. He found that as soon as the cell is committed to its transformation, overexpression of junk DNA begins to occur. When he explored this, he found that there were jumping genes that moved around the junk section of the genome. What Muotri found quite amazing was that the patterns of these jumping genes were distinct from one another and varied from cell to cell. “It was real,” he said, “you could see that every neuron had its own unique randomness during its formation.” When you think about the brain, Muotri’s research has paved the way for the use of IPSCs in you envision organization, order, and complexity. Randomness disease modeling. What may be more significant is the hope he has seems to be counterintuitive to the general functionality of the brain. provided for millions affected by Rett syndrome and other autism But when you examine the behavior of the brain, you can see that it spectrum disorders. Such breakthroughs in an area of disease where requires this instability in order for it to work. Flexibility is the key to many scientists seem to know very little about are inspiring. “While adapting to changing environments, and the human brain is a master my motivation lied in my pursuit to understand the human brain, I of this. The randomness in the development of the neurons leads could not be more pleased with the effect my research has had,” said to the uniqueness of each cell, which then leads to diversity and the Muotri. His laboratory exploded in the aftermath. Muotri and his team flexibility of the brain. are now looking at common molecular pathways Muotri then between idiopathic autism and Rett syndrome. The focused his he has received has also proved to be a research on to what Randomness seems to be attention valuable resource for his studies. The families of made the human counterintuitive to the general affected children began participating in sending cell brain so different samples to Muotri’s lab. It has become so popular from other animals’ functionality of the brain. But that he has created a tooth fairy collection service, brains. The answer which the parents send the lab their children’s he found lied in our when you examine the behavior infallen teeth, which provide usable cell samples, unique social ability. and the children receive a thank you note from the of the brain, you can see that it Studies found that “tooth fairy” in return. The program has helped humans are able requires this instability in order for him accumulate an entire library of cell samples to form roughly that he could use to molecularly compare different 150 memorable it to work. traits, such as levels of verbal and non-verbal skills, relationships with to one another. He also began comparing cells other humans, the gathered from Williams syndrome patients, in which the patient’s closest species being the chimpanzee, which can make less than 50 neuronal synapses are overexcited, to those of ASD patients and casual interactions. Using this information, he came to the conclusion seeing how one could achieve balance between the two. With so that the best way to understand these extremely complicated many possibilities and endless amount of potential, there are many interactions within the human brain would be to observe these more breakthroughs to come from Muotri and his lab. connections when they do not work properly. Muotri then decided that disease modeling would supply the best method for his studies and began his research. He targeted mental diseases, those common in ASD, which were considered to hinder social ability. Since Rett WRITTEN BY KEVIN PEREZ. Kevin Perez is a Human syndrome was thought to be caused by a mutation in neuronal Biology major and Philosophy minor from Earl Warren College. He development and Muotri had a background in genetics, it seemed will be graduating in 2014. like the obvious choice for his research. Muotri and his team set up their control to be a regular somatic cell from a normal adult, and the variables to be cells derived from Rett syndrome patients (RTT). After inducing pluripotency, 1. Marchetto, M. C., Carromeu, C., Acab, A., Yu, D., Yeo, G. W., Mu, Y., et al. Muotri and his team took these newly created stem cells and began (2010). A Model for Neural Development and Treatment of Rett Syndrome differentiating them into neurons. Their first discovery was that Using Human Induced Pluripotent Stem Cells. The Cell, 527-538.

the aftermath

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references

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AXONAL REGROWTH Sharon Vongvanith

staff writer

e have all walked into our kitchen before in the early morning with our eyes half open. The lights are not on, but the morning sunshine is beginning to peek through the curtains. And as we reach for something, we feel a throbbing sting on our hand. We have touched the bottom of a coffee machine or a pot on the stove, and we react with a painful, “Ouch! What was that?” This speedy reaction is due to our nervous system, the “executive” in charge of all the functions and intricacies of our organ systems. The nervous system accounts for all sensations, feelings, and the basic mechanisms and functions of a human being. The nervous system consists of three main components: a brain, a spinal cord, and a huge network of nerves that runs throughout the body. However, there are two divisions in the nervous system, the Central Nervous System (CNS) and the Peripheral Nervous system (PNS). The CNS consists of our brain and spinal cord, while the PNS consists of nerves and binds the CNS to the rest of the body. Both divisions of the nervous system allow the human body to carry out daily functions such as walking, talking, and breathing. They allow sensation and perception of our surrounding environment. Quite recently, Dr. Andrew Chisholm, Professor at the University of California, San Diego and Researcher of Developmental Biology and Neurobiology, and his team of biologists worked with Professor Yishi Jin, Professor of Neurobiology and Cellular and Molecular Medicine at University of California, San Diego. Information was found on the growth and regrowth of axons. An axon is one of three components of a neuron, or a nerve cell, that is vital to the nervous system. It conducts electrical impulses away from the nerve cell. The other two components of a nerve cell are the cell body and the dendrite. The cell body, or the soma, has similar organelles to other cells. A major difference between the body of a nerve cell and that of a normal cell is the soma’s inability to reproduce due to a sq.ucsd.edu

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FEATURES lack of a centriole. This is why damaged nerve cells can be concerning; they are difﬁcult to replace. The third major component of a nerve cell is the dendrite, which is similar to tree branches in appearance. Dendrites are attached to the cell body and communicate with other neuron impulses at their synapses, or the location where neurons “talk” to other cells.

NEURONS 101

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A high definition picture of a neuron in tissue culture. Diagram of a typical neuron; dendrites receive incoming signals and the action potential is propogated down on the axon. Myelin sheath speeds up the process by acting as an “insulator” for the axon, allowing the action potential to jump toward the terminal buttons of the axon end.

Photo courtesy of: www.en.wikipedia.org

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An artistic depiction of synaptic cleft activity. A neurotransmitter is released from the presynaptic terminal, crosses the synaptic cleft, and binds to receptors on the postsynaptic terminal. Single neuron with axons extending outwards; has been stained with antibodies to clearly show the cell body and nucleus.

http://www.fmengert.net

photo courtesy of mit.admissions.org

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Professor Jin’s Research Professor Yishi Jin’s lab works on understanding the signaling pathways of the nervous system and the abilities of adult neurons to regenerate. Her lab has approached axon regeneration in various ways, some of which include genetic, molecular, and cellular means. Ultimately, the goal of Jin’s lab is to use this research to apply to real life neurological disorders, and eventually, for repair. To better understand axon regeneration, it is important to first understand the relationship between a neuron and its partners. Neurons have growth cones, which are structures within the cell that guide the cell to its target. Many “motor neuron axon guidance” (max) genes, which control the way the axon locates its target, have been identified. For example, the max-1 gene controls the netrinsignaling pathway. Netrins are guidance molecules that aid the growth and function of neuronal circuits, the molecules that help neural and axonic pathfinding. Recently, Jin’s lab has published studies that have found how axons locate their target after an injury. To further our insight on axon regeneration, it is important to also understand the way neurons form synapses with its partners. Presynaptic terminals are where neurons control synaptic vesicles. The cytoarchitecture, or the cellular composition, of a presynaptic terminal is still being explored in depth using fluorescent markers to label the various parts of a presynaptic terminal to identify abnormalities. Jin’s studies have shown that there are new signaling molecules that reveal more information about presynaptic terminals. These molecules work through ubiquitin-mediated protein degration and GTPases. Understanding the formation of neural circuits during normal development may help us understand how injured neurons can recover and begin functioning normally.

Professor Chisholm’s Research Professor Chisholm’s lab focuses on the neuronal and epidermal tissue development and damage. C. elegans, a cheap and simple model organism, is used to understand these processes. Chisholm states, “The ability of axons to regrow after injury has been known for decades, yet axon regrowth mechanisms remain poorly understood owing in part to the lack of genetically tractable models. In collaboration with Jin’s lab, we have developed C. elegans as a genetically accessible model for axon regeneration.” Chisholm and his lab have found mechanisms for axon regeneration through the identification of genes that are expected to have influence on axon growth and development. It was found that “scientists detail their discoveries after an exhaustive two-year investigation of 654 genes suspected to be involved in regulating the growth of axons [where] researchers identified 70 genes that promote axon growth after injury and six more genes that repress the re-growth of axons.” Chisholm further states, “C. elegans neurons display robust regenerative responses that critically depend on the DLK-1 MAP kinase pathway and second messenger cascades.” The DLK-1 MAP kinase, or the Dual-Leucine zipper Kinase, promotes mRNA stability that influences axon regeneration. Simply put, the DLK-1 MAP kinase is an enzyme that helps mRNA enable the repair or regrowth of axons. An interview with Chisholm revealed more background information about this ongoing research project. Chisholm explains, “This work was started by a team of postdocs and graduate students sq.ucsd.edu

in 2004. It was motivated by pure curiosity. We were interested in the basic biological question of whether C. elegans axons could regrow after injury. Nobody had investigated this before, although it had been studied in many other kinds of animals. We were fortunate enough to collaborate with laser physicists at Stanford who could generate precise injuries in live C. elegans animals. That was the work published in Nature in 2004.” And while research on axon regeneration with C. elegans began out of pure curiosity, it is clear that Chisholm and Jin’s research will have positive impacts that will benefit the health of our society. For instance, axon damage is the reason why spinal cord injuries cause the loss of motor control and sensation. The physical injury causes the axon fibers to stretch and swell, making the axons more vulnerable to damage. While this damage is not immediate, it impairs axonal transport that transfers cellular components and other molecules from the cell body to the axon. The damage to the cell cytoskeletons, in severe injuries, changes the cell membrane. This results in an abnormal increase of ions like calcium. The cumulative effect is that neurons are not able to effectively communicate and transfer information among each other. Chisholm supports this by stating, “There could be very long term implications for treating human CNS injuries such as spinal cord injury, stroke, or optic nerve damage. These are significant problems in human health. Studies in mammals have led to insights but the focus is on a few molecules. We are taking the first systematic genetic approach; hopefully this will lead to better understanding of the basic biology as a precursor to better therapies.” As it stands now, it may take a while before scientists fully understand how to cure ailments of the nervous system, and those with major spinal cord-related injuries are left with no solution. However, if Chisholm’s and Jin’s research find how axon regeneration can be done more efficiently, then there may be answers to mending these injuries. The loss of sensation and motor control will not be permanent, and they will give hope to those who face such difficulties. These future findings will pave the way to discovering solutions to injuries such as these.

WRITTEN BY SHARON VONGVANITH. Sharon

Vongvanith is a high school senior at The Preuss School UCSD. She will graduate as valedictorian and attend a university in the east coast.

references 1. McDonald, K. (2011, September 21). UC San Diego Biologists Discover Genes That Repair Nerves After Injury. UC San Diego News Center. Retrieved from http:// ucsdnews.ucsd.edu/newsrel/science/2011_09neurons.asp 2. Axon Damage. (1996, September-October). National Institute of Neurological Disorders and Stroke. National Inst. Of Health. Retrieved from http://www.ninds. nih.gov/news_and_events/proceedings/sci_report.htm#Axon_damage 3. Chisholm, A. (2011, November 12). Professor: Sections of Cell and Developmental Biology and Neurobiology, UCSD. Division of Biological Sciences. Retrieved from http://biology.ucsd.edu/faculty/chisholm.html 4. The DLK-1 Kinase Promotes mRNA Stability and Local Translation in C. elegans Synapses and Axon Regeneration. (2009, September 4). Cell. Retrieved from http:// www.cell.com/abstract/S0092-8674(09)00723-5 5. Jin, Y. (2011, November 12) Professor: Section of Neurobiology Department of Cellular and Molecular Medicine, SOM Investigator, Howard Hughes Medical Institute, UCSD. Division of Biological Sciences. Retrieved from http://biology.ucsd. edu/faculty/jin.html 6. The Nervous System. (2011, November 12). Nature’s Best: The Human Body. Retrieved from http://library.thinkquest.org/2935/Natures_Best/index.html

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looking toward the future with

RESEARCH The Research section aims to highlight the driven undergraduate researchers at UC San Diego, whose work constantly expand the boundaries of science.

SALTMAN QUARTERLY â&#x20AC;˘ VOL 9

sq.ucsd.edu

RESEARCH

sq.ucsd.edu

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RESEARCH

Is Algal Growth on Acropora Coral Limited by Small Grazers? Jordan Arce

The effects small grazers have on algal growth in hard to reach areas of Acropora coral were studied on Heron Island in the Great Barrier Reef. Fish were observed on the reef to assess whether large herbivorous fish had more of an effect on algal growth on the outside than the inside of coral in the hard to reach places. Xanthid Leptodius crabs were collected and placed in tanks alongside pieces of algae-covered Acropora coral that represented the inside of a coral colony to observe their cheliped bite and feeding rates throughout the day. Coral weight, detritus weight, and dry algal weight were measured from control and treatment tanks to determine the crab’s influence on the coral piece. On average there were 61.8% less bites taken on the outside of Acropora by small fish than large fish, and 92.1% less bites taken on the inside than outside of Acropora by large fish. The average wet weight of coral in control bottles was significantly higher by 7.55% than that of coral in bottles with crabs after four days of treatment. On average there was more algal biomass in the bottles without crabs; 90.2% more Lobophora, 40.8% more turf algae, and 1.78% more Hypnea. On average there was 85.7% less detritus observed in the control bottles than in bottles with crabs. Cheliped bite means for afternoon and night were considerably higher than that of the morning. Feeding rate means steadily increased from morning to night. It was examined that large herbivorous fish prefer to bite out on the outside of Acropora, and that xanthid crabs limit algae in hard to reach areas of Acropora coral. Keywords: Acropora • algal growth • algal dominance • herbivorous fish • grazing • limiting factor • xanthid • coral reef

Introduction

Coral reefs are important structures in the world’s oceans because they contain high levels of biodiversity and act as nutrient and energy oases in oceanic deserts, providing a home for fish, molluscs, crustaceans, cnidarians, and other invertebrates. They also provide many ecosystem services to the global economy worth more than $375 billion each year. Algal growth on coral is a common feature, but its effect on the coral can vary. Healthy reef communities can contain hundreds of species of macroalgae as a natural part of the ecosystem, but coral dominance can start to decrease when algae become too abundant.1 Phase-shifts from coral to macroalgal dominance have been observed in the Pacific, Indian, and Caribbean Oceans.2 Anthropogenic effects can explain these shifts. For example, algal growth has been associated with increased nutrient input.1,3 Simultaneously, overfishing reduces grazing by herbivorous fish that controls algal growth.4,5 Algae are important parts of the reef system because they supply an entire trophic chain, including many small invertebrates and a huge variety of herbivorous fish. Invertebrates that graze on the outside of coral have a lesser effect on algal growth than herbivorous fish that graze similarly.6 Algae will quickly colonize vacant coral substrate and may even be particularly abundant in holes or crevices where herbivores cannot easily reach.1 Acropora is a branching coral in the Great Barrier Reef of Australia that contains many crevices and holes that algae can monopolize.

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Often, however, Acropora shows little algal dominance. Although herbivorous fish do feed on algae growing on the outside, invertebrates, such as xanthid crabs and possibly smaller fish, may be limiting algae in the hard to reach places on Acropora, preventing it from dominating the coral colony. This present study is focused on determining the effects small grazers have on algal growth in hard-to-reach areas of Acropora coral on Heron Island in the Great Barrier Reef. Fish were observed on the reef to assess whether large herbivorous species had more of an effect on algal growth on the outside than the less accessible parts of the coral. Xanthid Leptodius crabs were collected and placed in tanks alongside pieces of algae-covered Acropora coral that represented the inside of a coral colony to observe their cheliped bite and feeding rates throughout the day. Coral weight, detritus weight, and dry algal weight were measured from the control and treatment tanks to determine the crab’s influence on the coral piece. If crabs can limit algae by feeding where fish cannot, larger fish are predicted to take more bites from outside of the Acropora than inside while Xanthid crabs will feed in hard-to-reach areas of Acropora coral.

Materials and Methods

Fish Observations Observations of ﬁsh herbivory on Acropora were performed by snorkelling for two hours in the Heron Island harbour (23° 26’ 31S, 151° 54’ 45 E) in high tide during

the day over the course of three days. The number of bites taken on Acropora were counted and separated by location of bite (inside or outside of coral) and size of fish (greater than 10 cm as large and less than 10 cm as small) as well as family (Blennidae, Scaridae, Acanthuridae, Pomocentridae, and Siganidae were targeted as they encompass the main herbivorous fish on the reef). Crab and Coral Collection Xanthid crabs were collected during the night low tide from Acropora stands off the research beach (23° 26’ 36 S, 151° 54’ 45 E) and put in a holding tank for two days. Dead coral with algal growth were collected from the research beach. Those with Epilithic Algal Matrix, Lobophora, Hypnea, and red and brown turf algae cover were selected for the experiment, and those with Sargassum, Colpemenia, Hydroclathera, and red encrusting algae were excluded as did they did not represent commonly grazed algae on Acropora. Twelve coral pieces were formed by fragments of coral that were collected and tied together or by broken-off coral pieces in the same relative shape (several branches off a coral stem) and sized to fit inside a two-liter bottle without touching the sides. Detritus and loose algae were rinsed off the coral pieces before their wet weights were measured. Design Two-liter plastic bottles were used as observation tanks for coral pieces and crabs. Each bottle’s base was cut off, and a nylon sq.ucsd.edu

RESEARCH mesh cut from stocking was secured between the head of the bottle, where it begins to curve into the spout, and the rest of the bottle. With the bottles upside-down, nylon acts as a mesh for catching detritus as well as for keeping crabs from resting into the bottle spout. Bottle caps were twisted shut. Holes were punctured around the bottle above the mesh to allow water flow. Each coral piece was suspended by a string in individual bottles from a horizontal skewer placed near the open base of the bottle. Coral pieces were suspended close enough to the mesh for a crab to climb onto them. Coral branches were oriented up towards the open base of the bottle. Twelve bottles with coral pieces inside were suspended in an open aquarium tank with constant seawater flow, with the spout end pointing down. Each bottle’s location in the tank was chosen randomly. Six Xanthid crabs were randomly placed in a bottle. Clear cellophane was used to cover the open base of each bottle to prevent crab escape but was punctured with small holes to allow water flow. Experiment The study’s treatment consisted of six bottles with crabs, and the control consisted of six bottles without crabs. Observations were not started until one day after the experiment was set up to allow crabs to acclimate to their new environment. Observations consisted of counting cheliped bites, with one bite defined by observing a crab retract its chelipeds from the coral to its mouth. Total number of cheliped bites for each crab was observed for 90 minutes in

Figure 1. Histogram displaying mean amount of bites taken by large and small fish on the inside and outside of coral colonies, plus or minus standard error. There is a significant difference between number of bites taken by big and small fish (p=0.006). There are a greater number of bites taken outside of the coral colony than inside by large fish (p=0.005), and there is no significant difference in amount of bites taken inside and outside by small fish (p=0.954).

the morning (defined between 9am-12pm), afternoon (2-5pm), and night (8-11pm) for three days to observe feeding behaviour. Observations at night were made under a red light to prevent disturbances to their nocturnal activity. Total number of fiveminute intervals that each crab was feeding was also observed, with an interval defined as a period of two minutes. After three days of crab observations, coral pieces were taken out, and their wet weights were measured. Algal biomass was measured by scraping off algae from five

Figure 2. Histogram displaying average wet weight of coral after experiment, plus or minus standard error. There is a significant difference between wet weight of control and of treatment (p=0.00641). Average wet weight of control coral is significantly higher than wet weight of treatment coral. sq.ucsd.edu

random two-by-two centimeter subsamples of each replicate coral piece. Algae was separated by species and weighed after being dried in an oven. Each bottle was taken out of the water slowly to allow floating particles to settle onto the mesh. With bottle cap slowly taken off, remaining water was allowed to flow out through the spout onto filter paper to collect detritus. An additional 200mL of seawater was used to rinse out particles resting on the sides of the bottles and on the mesh onto the filter paper for each bottle. 200mL of seawater was used for each bottle for consistency and to account for added weight of saltwater ions. Filter paper for all bottles were dried in an oven and weighed, with the added saltwater weight subtracted to give the weight of just the detritus. Statistical Analysis Ten small fish and ten large fish observations were randomly chosen for comparing number of inside and outside bites from the pool of data. Significances between small and large fish, and inside and outside bites were tested through twofactor ANOVA using R statistical software. Significance of cheliped bites per interval between the morning, afternoon, and night observation periods were tested through a three-factor ANOVA. Significance of proportion of intervals of crab feeding between morning, afternoon, and night observation periods was tested through a

VOL 9 • SALTMAN QUARTERLY

RESEARCH morning to night (Fig. 6). Additional behavioral observations Fish herbivory during low tide observed by snorkelling was relatively low compared to high tide, and most ﬁsh, such as Pomacentrids and Blennies, were more concerned with keeping territory than feeding while Scarids did not seem interested in feeding as they continually swam past coral stands. Generally, most crabs were more active feeding and moving around the coral at night and were able to eat algae from anywhere on the coral piece, regardless of how many crevices, holes, or branches it had.

Discussion

Figure 3. Histogram of average dry weight of algal biomass of different species between treatment and control, plus or minus standard error. No significant difference of algal biomass was found between treatment and control (Lobophora: p=0.64244, Turf: p=0.39747, Hypnea: p=0.85706).

three-factor ANOVA. Significance of wet weights between control and treatment coral after crab experiment was tested through a one-factor ANCOVA, as there was a strong linear relationship between the before and after wet weights of each coral piece. Significance of dry weights between control and treatment coral after the crab experiment was tested through a three-factor ANOVA. Significance of the detritus weight between control and treated bottles was tested through a two-tailed t-test.

Results

A total of 122 inside bites and 299 outside fish bites of algae were observed on coral in the harbor during high tide over three days. All Siganids and Scarids were large, all Pomecentrids and Blennies were small, and most of the Acanthruids were large. 29% of the total observed bites were inside, and 71% were outside. Of the outside bites, 9% were taken by Siganids, 52% by Scarids, 9.4% by Pomocentrids, 23% by Blennies, and 8% by Acanthurids. Of the inside bites, 12% were taken by Scarids, 51% by Blennies, 5% by Acanthurids, and 33% by Pomocentrids. Approximately the same number of bites were taken on the outside and inside of Acropora by small fish, making their comparison statistically insignificant. On average, fewer bites were taken on the outside of Acropora by small fish than large fish, while fewer bites were taken on the inside than outside of Acropora by large fish (Fig. 1). After four days of treatment, the average wet weight of coral in the control

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bottles was greater than that of coral in bottles with crabs (Fig. 2). Dry algal biomass in treatment bottles was less than that in control bottles by small margins (Fig 3). On average, less detritus was observed in the control bottles than in the bottles with crabs (Fig. 4). The average number of crab cheliped bites of per interval of time for afternoon and night were considerably higher than that of the morning (Fig. 5). The proportions of intervals feeding steadily increased from

Larger herbivorous fish prefer to feed on the outside of Acropora. Xanthid crabs limit algae in hard to reach areas of Acropora coral. These findings may be able to explain how algae are limited on Acropora in a coral reef system of the Great Barrier Reef although they may not apply to all systems. For example, five herbivorous fish families were observed in this study, but in other systems only one family or species can dominate herbivory on a given reef, suggesting that the vulnerability of different reefs to algal dominance may depend more on fish species than fish size.2 Additional studies have observed a greater abundance of herbivorous fish in areas where algae has already been greatly diminished, implying that factors in addition to fish herbivory may be more important than the amount of algae present.8 Besides algal growth, sedimentation

Figure 4. Histogram of average weight of detritus from control and treated bottles, plus or minus standard error. There is a significant difference between control and treated bottles (p=0.009). A significantly higher average weight of detritus was observed in the treated bottles. sq.ucsd.edu

RESEARCH

Figure 5. Histogram of average number of cheliped bites during different periods of time throughout the day, plus or minus standard error. No significant difference was found between average number of cheliped bites among the different periods of time observed (p=0.833).

can also decrease coral health and represents one of the most common causes of coral reef degradation.7 Interestingly, one study concluded that certain crabs were successful at removing grain sizes from the coral and reviving the health of the coral colony from sedimentation effects.8 Xanthid crabs thus may not only prevent algal dominance but decrease sedimentation effects on coral reefs. Seaweed cover has been observed to be an important limiting factor to temperate reefs, another system in which grazing may be a factor inﬂuencing coral health.9

Josh Osias, Tina Hannouche, Lauren Smith, and Sonya Benjamin, as well as Heron Island Research Station staff.

References

1. Vroom, P.S., Page, K.N., Kenyon, J.C., Brainard, R.E. (2006). Algae-Dominated Reefs. American Scientist, 94, 430-437. 2. Cvitanovic, C., Bellwood, D.R. (2009). Local variation in herbivore feeding activity

on an inshore reef of the GBR. Coral Reefs. 28, 127-133. 3. Williams, I.D., Polunin, N.V.C. (2001). Large-Scale associations between macroalgal cover and grazer biomass on mid-depths reefs in the Caribbean. Coral Reefs, 19, 358366. 4. Russ, G.R. (2003). Grazer biomass correlates more strongly with production than with biomass of algal turfs on a coral reef. Coral Reefs, 22, 64-67. 5. Polunin, N.V.C., Klumpp, D.W. (1992). Algal food supply and grazer demand in a very productive coral-reef zone. Coral Reefs, 164, 1-15. 6. Rivera, E.C., Paul, V.J. (2006). Feeding by coral reef mesograzers: algae or cyanobacteria? Coral Reefs, 25, 617-627. 7. Stewart, H.L., Holbrook, S.J., Schmitt, R.J., Brooks, A.J. (2006). Symbiotic crabs maintain coral health by clearing sediments. Coral Reefs, 25, 609-615. 8. McClanahan, T.R., Henrick, V., Rodrigues, M.J., Polunin, N.V.C. (1999). Coral Reefs, 18, 195-203. 9. Willer, M.W., Hay, M.E. (1996). CoralSeaweed-Grazer-Nutrient Interactions on Temperate Reefs. Ecological Monographs, 66, 323-344.

About the Author

Jordan Arce is an Ecology, Behavior, Evolution major from Sixth College. He graduated in 2011.

Conclusion

Results of my study are consistent with the inﬂuence of crabs on limiting algal growth. Further research should examine other important invertebrates on Acropora, such as Saron shrimp, and invertebrate algal species preference. Nutrient effects should also be studied and compared against grazing affects in coral reef systems. Other corals such as Pocillopora also can be studied in terms of factors that prevent algal dominance. The design of the bottles in my study was preliminary and may have been among the study’s limitations. Future containers should focus on sealing the bottles at the spot and possibly implementing a valve with which to easily drain and collect detritus.

Acknowledgements

Acknowledgements go to advisors Dr. Ian Tibbetts and Renee Rossini and colleagues sq.ucsd.edu

Figure 6. Histogram of proportion of intervals crabs were foraging to total amount of intervals observed per time of day, plus or minus standard error. There was no significant difference between proportion of intervals foraging among morning, afternoon, and night (p=0.216). VOL 9 • SALTMAN QUARTERLY

RESEARCH

Effects of Habitat Complexity and Fishing Pressure on Moray Eels (Muraenidae) Along Santa Elena Peninsula, Costa Rica Eric Garcia

Complex habitats such as coral and rocky reefs are very important in oceans as they are thought to increase overall fish abundances. However, such habitats often serve as refuge for prey fish and, as a consequence, reduce the rate at which marine predators successfully catch prey. This may negatively impact the diversity and abundance of predator species that live in complex habitats. Moray eels (Muraenidae) are predators that are largely found in complex habitats but, unlike most other marine predators, they possess adaptations for living in complex habitats such as their lack of pectoral fins and their ability to swim backwards. These characteristics in complex habitats may give morays an advantage over their prey. Yet, the effects of habitat complexity on morays eels have not been extensively studied before and thus require further investigation. In this study, I analyze the effects of habitat complexity on moray diversity and abundance by comparing moray populations of offshore islands and coastal sites that presente diverse habitat complexities along the north and west coasts of the Santa Elena peninsula, Costa Rica. I further study this relationship by contrasting observations obtained in a previous study conducted at Bajo Rojo, a complex rocky reef offshore of Cuajiniquil, with new observations from the same study site. Lastly, I evaluate the effects of fishing on moray communities by comparing those dwelling in Bajo Rojo, where fishing is allowed, to those inhabiting the complex coral reefs of the protected waters of Islas Murciélago. I did not observe any individuals in sites with low complexity, and there were only few moray species in habitats with intermediate complexity. In contrast, the highly complex Bajo Rojo and Islas Murciélago shared the highest species richness, strongly suggesting that habitat complexity enhances moray diversity. Results further reveal that highly complex habitats are capable of maintaining high levels of moray diversity after a one-year lapse. Finally, fishing appears to have no effect on moray abundance and diversity but obtained data suggests that it might reduce average moray body size. Keywords: Muraedinae • fishing pressure • habitat complexity • species composition • population dynamics

Introduction

Habitat Complexity refers to the physical arrangement and accumulation of objects in space. In the ocean, coral and rocky reefs are typically some of the most complex habitats as they often present large structures underwater such as walls and boulders. Some of the characteristics of these reefs include the accumulation of rocks of different sizes, systems of holes and tunnels in the reef framework, cracks and parallel ledges along walls, spaces between two adjacent boulders, etc. Such structural features provide refuge and many other services to fish. Menard affirms that habitat complexity increases fish abundances, and Bell states that it greatly influences local fish abundance and diversity.2,6 Yet complexity is also thought to reduce predation by providing more refuges for prey and/or by reducing the rates in which prey and predators encounter each other.8 In other words, complex habitats often serve as sanctuaries and nurseries for prey species. Thus, complexity might negatively affect marine predator populations. However, moral eels, unlike other marine predators, have adaptations for a complex habitatdwelling lifestyle such as the lack of pectoral and pelvic fins, which allow them to navigate easily through narrow spaces and live within reef frameworks. In addition, morays also have evolved the ability to swim both forwards and backwards, allowing reverse movement into refuges.3,5 Such abilities and adaptations may give morays an advantage over their prey in marine complex habitats. Furthermore, highly complex habitats also provide morays with refuge as they

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are also sometimes preyed upon by other eels or bigger predators such as sharks.4 Consequently, the relationship between habitat complexity and moray diversity and abundances is not obvious given that habitat complexity can affect morays (1) negatively by reducing their prey-encountering rate and (2) positively by increasing refuge availability. In this study, I intend to determine whether complexity increases, decreases, or has no effect on moray diversity and abundances. I am also interested in resolving whether moray populations can remain stable in highly complex habitats. Thus, I document changes on moray richness, abundances, and body size in the two studied sites with high habitat complexity levels: Bajo Rojo, a rocky reef formed by uplifted seafloor with variable rocky substrates at Bahía Cuajiniquil, and Islas Murciélago, an archipelago exhibiting extensive coral reefs west of Santa Elena peninsula. I perform this analysis by comparing observations from Nicholson, who surveyed moray populations from the 7th to the 19th of May 2008 at Bajo Rojo and Islas Murciélago to my own observations obtained one year later at the same sites.9 In addition, the fishing pressure on morays at Bahía Cuajiniquil and Santa Elena peninsula is also ambiguous. Morays are not commercially fished; however, large morays are sometimes harvested so their flesh can be used as bait for other fishing practices. Likewise, fishermen that feel threatened in their presence often kill large morays.7 Thus, I also evaluate the effects of fishing on moray diversity, abundances and body size by comparing populations at the same

two complex habitats, Bajo Rojo, which is a fishing site commonly visited by local fishermen, and Islas Murciélago, a Marine Protected Area (MPA) where fishing is prohibited. I then ask whether fishermen may exert any effects on moray species richness, abundances and body size.

Materials and Methods

I identified each observed moray according to the morphological features presented by Allen & Robertson.1 If identification was not possible from the surface, I submerged myself to have a closer look to more easily identify the eel. Pictures from all the observed moray species are illustrated in Figure 1. I further classified every observed eel into three distinct body sizes: small (<0.6m), medium (0.6-1.2m), and large (>1.2m). Body size was estimated by sighting the whole body or the head diameter (in case whole body was not visible). I conducted my research during late April and early May 2009 in the north and west coasts of the Santa Elena peninsula, Guanacaste, Costa Rica (Fig. 2). On land, this peninsula is part of the Santa Rosa National Park, which is dominated by tropical dry forest. The north and west coasts consisted of mostly rocky shores and few sandy beaches. These coasts along with the near offshore islands offered the ideal settings for this study as they presented coastal sites with a diverse habitat complexity gradient (Table 1). I visited every site only once except for Bajo Rojo where I snorkeled on several occasions. I conducted the majority of my sq.ucsd.edu

RESEARCH Date visited

Site

Type of substrate

Complexity level

Total # of species observed

Average Species observed /30 min

Rate of observation /30 min

Sand banks with April 26

Isla David

scattered rocks Sand banks and

May 12

Bahía Coco

Bahía Coquito

Low

to rocky coast both covered in algae Intermediate Sand banks and

May 12

Low

to rocky coast both covered in algae Intermediate Low to Rocky coast covered in algae Intermediate

May 12

Bahía Dante

May 6

Bahía Thomas (West) Rocky coast

Intermediate

May 5

Dock/ Muelle

Rocky coast

Intermediate

3.5

May 7

Bahía Thomas (East) Rocky coast

Intermediate

3.5

May 9

Isla Phytaya

Rocky edge

Intermediate

May 9

Bahía Lucas

Rocky coast

Intermediate

1.5

May 15

Islas lago

1.25

2.5

4.14

Murcié- Sand banks and coral reefs High

May 5 to 16 Bajo Rojo

Rocky reefs

High

Table 1. Site information and dates in 2009 that each survey was performed. The total number of species, the number of species observed per 30 minutes, and the rate at which individuals were observed per 30 minutes of snorkel effort are also given.

ﬁeldwork at Bajo Rojo due to its proximity to our housing in Cuajiniquil and, for that reason, in contrast to most other sites along the Santa Elena Peninsula, it contained important habitat complexity. Only Islas Murciélago offered similar complexity but this site is considerably further away from Cuajiniquil; hence, I visited it only once, as with all the other sites. Data was collected through snorkeling surveys of approximately 60-90 minute interval per day over 13 non-consecutive sq.ucsd.edu

days (Table 1). All surveys were conducted starting approximately at noon. The total snorkeling effort was not equal in each site; therefore, for all statistical analysis I compared moray populations by the rate at which individuals and the number of species were observed instead of total observations per site (species observed/30min and observations/30min, respectively). Surveys at Bajo Rojo were done following the same procedure as Nicholson, I started snorkeling at the northwest point on the leeward or

south side of the reef swimming in a zigzag fashion and eastbound direction.9 Such methodology enabled careful eel inspection in the reef and minimized the possibility of counting the same moray multiple occasions during a particular snorkeling session. On every other site, I snorkeled a distance of approximately 100 meters in a linear manner along the mainland or around it in the case of Isla Phytaya. The survey at Isla David was conducted on the west side of the island only. I inspected areas under rocks at least once every 15 to 20 meters for every survey. Habitats of all 11 studied sites included sand banks, rocky coast, and rocky and coral reefs. The complexity of each site was classified according the variety and/or the number of structural features available to fish. Sites where there were only sand banks and/or only few rocks were classified as having low complexity; sites offering plenty of rocks or other small structures covering approximately 50% of the seafloor were considered to be low to intermediately complex; habitats where most of the seafloor was covered by rocks but lacked large boulders, walls, or reefs were categorized as having intermediate complexity; and sites with extensive resources, rocks, and large frameworks and were group as highly complex habitats. Dates as well as complete site information are given in Table 1. For the habitat complexity analysis, I compared the number of moray sightings and diversity between all the different sites. For the fishing pressure evaluation, I compared and contrasted my observations from Bajo Rojo and Islas Murciélago to those of Nicholson.9 I conducted this part of the study at these two sites since they were the best suited for this analysis given their very similar high complexity and completely opposite fishing protection. Any discrepancy in the moray populations can be attributed to the fishing protection differences and not habitat complexity. Additionally, I considered that ocean tides and seawater temperature could potentially influence moray observations as the tidal range exposed the upper part of reefs and currents of cold water were not unusual in the area.7 Thus, I analyzed any effects that these had on my data collection at Bajo Rojo, where I performed most of the surveys and the only site I visited in multiple occasions. Tides were classified as high if less than 1 meter of the reef was expose to the air; as medium if 1-2 meters were uncovered; and as low if 2 or more meters were visible. I directly measured the seawater temperature with a thermometer at Bajo Rojo on May 11th, 14th, 15th, and 16th.

Results

I observed a total of 128 individuals from 8 different moray species (Fig. 1). I found a statistically strong positive relationship between habitat complexity and moray diversity (correlation coefﬁcient r=0.84, where r=1.0 represents a perfect positive relationship). Speciﬁcally, I documented VOL 9 • SALTMAN QUARTERLY

RESEARCH high species richness at highly complex sites and low richness at sites with low complexity (Table 1). Furthermore, I encountered morays at different rates at each site, with the exception of the sites where morays were not observed. The relationship between complexity and the rate of observations was also statistically positive (correlation coefficient r=0.69). This was obvious in the field as the rate at which I observed morays increased at each site with higher complexity than the previous one. I did not observe any moray individuals in sites with low complexity levels. Every other site presented a unique eel composition (Fig. 3). Small and medium eels such as the jewel, starry, and palenose were the most abundant throughout the peninsula and across the complexity gradient. Instead, large moray such as the pandemic green, hourglass, and zebra were rarely seen at intermediate complex sites. The majority of these morays were observed at the highly complex coral reefs of Islas Murciélago. During the total snorkeling effort at Bajo Rojo, I observed a total 79 morays and witnessed the highest diversity of every studied site (Fig. 4). This observational set was very similar to the data collected by Nicholson one year before this study.9 Moray compositions in both years were similar with the exception that Argus morays were not observed in 2008 and that I did not find any individuals of two-holes (Uropterygius versutus) or slenderjaw in Bajo Rojo as in 2008. Nonetheless, there was no statistically significant difference between the number of sightings and species composition between 2008 and 2009 (X2= 8.62, df=8, 0.5 >p>0.1). Corresponding to body size, Islas Murciélago was still dominated by large morays whereas in Bajo Rojo, morays appeared to have shifted to a bigger average body size in comparison to 2008 (Fig. 5). The great majority of my sightings in Bajo Rojo were of small and medium morays, but I also observed few large individuals. These observations of moray body size at Bajo Rojo were statistically different from 2008 (X2 = 10.3, df=2, p<0.01). On the other hand, large eels, pandemic green in majority, were clearly dominant in Islas Murciélago (the only protected site) as they accounted for half of my observations. There, I observed medium-body morays as the second most abundant, and small individuals were uncommon. Sightings from Islas Murciélago did not statistically differ between 2008 and 2009 (X2 = 4.09, df=2, p>0.1). In terms of external factors, I found that ocean tides did not significantly influence moray sightings at Bajo Rojo (X2 =0.28, df=2, p>0.5). Finally, I documented a positive relationship between seawater temperature and the total moray observations at Bajo Rojo (correlation coefficient r=0.94).

Discussion

Effects of Habitat Complexity on Moray Populations I encountered different species

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Figure 1. Species encountered on this study (2009). Identification based on morphological features. Palenose, green, and slenderjaw moray pictures obtained from Eric Nicholson.

compositions at each of the studied sites. Given the strong positive relationship between complexity and the number of species observed, this variance could be attributed to differences in structural complexity and resource availability of each site. For instance, I found the highest species diversity at Bajo Rojo and Islas Murciélago. This high species richness most probably originated from the high complexity of the rocky reefs in Bajo Rojo and coral reefs in Islas Murciélago, where distinct refuges and other resources are continuously abundant allowing many ecologically distinctive moray species to co-exist. The following less complex sites illustrated a clear drop of

moray diversity of least 50% although most of the time this disparity was much greater. Consistently, I did not see any morays at any of the studied sites with low or low to intermediate complexity. This was most probably due to the fact that these sites only offered areas covered with algae or large sand banks with few rocks but no other type of refuge or resources. Results, then, strongly suggested that habitat complexity enhanced moray diversity. Moreover, the similarity between my data and that of Nicholson illustrated that highly complex habitats such as Bajo Rojo were capable of maintaining high levels of moray diversity through long periods sq.ucsd.edu

RESEARCH

Figure 2. Map of Santa Elena peninsula displaying the studied sites.

of time.9 This was most likely due to its structural complexity which translates into a continuous availability of distinct refuges and other resources. The relationship between habitat complexity and moray abundances did not become as clear as with diversity (correlation coefﬁcient was lower). Nonetheless, results suggested that complexity might also increase abundances as I also documented a positive relationship between complexity and the rate at which I observed the eels. Here, I surveyed the highest rate of sightings at Bajo Rojo and unexpectedly, a relative low frequency of sightings at Islas Murciélago. This last was surprising given the high habitat complexity of the archipelago. However, this low rate of sightings can be explained by the fact that Islas Murciélago, although isolated from mainland, had extensive areas of highly complex coral reefs and rocky shores along the series of near islands allowing morays to navigate longer distances and achieved more spread distributions. In contrast, Bajo Rojo was to some extent isolated from near habitats such as islands and mainland, limiting moray dispersal and possibly concentrating morays in its surroundings. Therefore, it was easier to observe morays at Bajo Rojo. In order to see the same amount of morays that I observed at Bajo Rojo, I would most likely have to snorkel for longer distances and/or for longer periods of time at Islas Murciélago. Despite of this, if the sighting rate of Islas Murciélago was treated as an outlier and excluded from the analysis, given the mentioned site characteristics, then, the relationship between habitat complexity and rate of sightings would follow a clear, strong, positive trend. Although moray sightings do not illustrate absolute abundances, they are representative of the relative abundances of the different moray species dwelling at the studied sites. Thus, habitat complexity may indeed increase moray abundances sq.ucsd.edu

as well. Yet, this relationship needs further investigation. Effects of Fishing Pressure on Moray Populations Clearly, fishing pressure had no effect on moray diversity (at least on these sites) as I documented high levels of diversity at both the non-protected Bajo Rojo and the protected Islas Murciélago. I do not make any conclusions about the relationship between fishing and moray abundances due to the relative low rate of sightings in the protected islands, which could have been influenced by other factors that are beyond the scope of this study, such as proximity to the open ocean, changes in currents, etc. Nevertheless, fishing relief was found to influence average body size in moray populations. The data suggested that morays at Bajo Rojo might be shifting towards larger body sizes, which was most probably a product of fisherman harvesting or killing less frequently than previous years. Yet, Bajo Rojo was still largely dominated by small moray eels. In both years, jewel, hourglass and starry morays were observed more frequently respectively. The relative higher jewel sightings may be explained by their behavior. Jewels appeared to be the less disturbed by our presence, less shy, even at close proximity. Other species tended to hide or flee when they saw us or when we got closer. On the other hand, large morays composed the 50% of my total sightings at Islas Murciélago. Results, hence, corroborated the significant higher proportions of larger morays at Islas Murciélago than at Bajo Rojo and reaffirmed one of the positive outcomes that marine protected areas offer to organisms that inhabit them. These observations also indicated that moray size was more influenced by protection from fisherman than by habitat complexity. Physical Factors The fact that I found no significant effect

of tides on the total observations of morays was most likely due to the physical size of Bajo Rojo. During the snorkeling sessions at low tide, the upper sections of the reef were exposed, but there were still a lot of rocky substrates underwater where morays could retreat. In this manner, morays still had substantial shelter in which they may dwell and were not forced to ﬂee to deeper and less complex habitats away from Bajo Rojo. On the other hand, data illustrated a positive relationship between seawater temperature and the total number of sightings at Bajo Rojo. In a particular day when there was a sudden 2-3ºC drop in seawater temperature relative to the previous days, I counted only 3 morays in comparison to 8 or 10 individuals from the previous and the day after. This was no surprise to our boat manager Minor Lara, who mentioned that in some rare occasions when sea temperature suddenly dropped several Celsius degrees, numerous ﬁsh including morays emerged dead. Although observations clearly supported a positive relationship between temperature and moray sightings, the analyzed data was limited as I measured the temperature of only 4 days. Therefore, additional studies are needed to determine the relationship between seawater temperature and moray observations and possibly abundances. In either case, seawater temperature most

Muelle

Bahia Tomas East

Isla Phytaya

Bahia Lucas

Islas Murcielago

Bajo Rojo

Figure 3. Illustration and comparison of moray composition, number of observations, and percentage of each species that occurred at each particular site. Bahía Thomas (west) was not included as only one slenderjaw individual was observed. Isla David and Bahías Coco, Coquito, and Dante were also excluded, as no morays were observed there. VOL 9 • SALTMAN QUARTERLY

RESEARCH an unforgettable experience. Finally and very importantly, thanks to all the people that gave rides in their motorcycles around Costa Rica. Pura Vida, chato mae! Mae! A jalar la panga! Chato mae!

References

Figure 4. Comparison between the total number of morays observed at Bajo Rojo, per species common name, during 2008 (Nicholson’s data) and 2009 (this study).

sightings, the analyzed data was limited as I measured the temperature of only 4 days. Therefore, additional studies are needed to determine the relationship between seawater temperature and moray observations and possibly abundances. In either case, seawater temperature most probably did not influence my results as it remained fairly constant during the great majority of the time conducting fieldwork. Significance of the findings This study has demonstrated that complex habitats yielded high moray diversity and might be positively linked to high abundances as well. Furthermore, results also showed that complex habitats are capable of maintaining high levels of moray diversity through long periods of time. This has particular importance since having high levels of diversity of predators such morays may help preserving the overall diversity and health of the entire fish community living in these complex habitats. These findings have implications for Marine Protected Areas as well. The observed larger moray body sizes at Islas Murciélago illustrated one of the benefits that MPAs offer to fish. Large body sizes may have translated into higher survival rates and higher reproductive success, contributing to health and growth of moray populations. Additionally, the findings referring to the effects of habitat complexity on moray populations were also valuable when designing MPAs. Explicitly, MPA architects needed to place careful attention to habitat complexity since as suggested by others and this study, complexity could yield and maintain high levels of diversity and abundances of prey species and predators, such moray eels. MPAs architects should

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then make an effort to include the maximum complexity possible inside the protected areas.

Acknowledgements

I would like to express my gratitude to Frank Joyce and Federico Chinchilla for their support throughout the entire EAP program. Thank you to all my snorkeling buddies that got wet with me. Thank you to captain Minor Lara who made a new adventure of every trip in his boat. Also, thank you to my reviewers for polishing my paper, especially to Dr. Heather Henter for her comments and help on the manuscript. Of course… thank you to all other students that made the Tropical Biology and Conservation Program

1. Allen, G. and Robertson, R. (1994). Fishes of the Tropical Eastern Pacific. University of Hawaii Press, Honolulu, Hawaii. 53-55. 2. Bell, S.S., McCoy, E.D., Mushinsky, H.R. (1991). Habitat structure: the physical arrangement of objects in space. Chapman and Hall, London 3. Gilbert, M., Rasmussen, J., Kramer, D. (2005). Estimating the density and biomass of moray eels (Muraenidae) using a modified visual census method for hole-dwelling reef fauna. Environmental Biology of Fishes, 73, 415426. 4. MacArthur, R.H., Levins, R. (1967). The limiting similarity, convergence, and divergence of coexisting species. Am Nat, 101, 377–385. 5. Mehta, R. Wainwright, P. (2008). Funtional Morphology of the Pharyngeal Jaw Apparatus in Moray Eels. Journal of Morphology, 269, 604–619. 6. Menard, A., Tugeon, K., Kramer, D. (2008). Selection of diurnal refuges by the nocturnal squirrelfish, Holocentrus rufus. Environ Biol Fish, 82, 59-70. 7. Minor, L. Experienced fisherman and dive master that have accumulated local knowledge throughout the many years he has lived in Cuajiniquil. 8. Murdoch, W.W., Oaten, A. (1975). Predation and population stability. Adv Ecol Res, 9, 1–132. 9. Nicholson, E. (2008). Diurnal Refuges of Moray Eels (Muraenidae) in the Bahía Cuajiniquil, Costa Rica. EAP Tropical Biology and Conservation Program, Spring 2008.

About the Author

Eric Garcia is an Ecology, Behavior, Evolution major from Earl Warren College. He graduated in 2010.

Figure 5. Comparison between moray body size observed in 2008 (Nicholson, 2008) and 2009 (this study) at Bajo Rojo and Islas Murciélago. sq.ucsd.edu

RESEARCH

The Exogenous and Endogenous Mechanisms Inﬂuencing the Locomotive Activity and Burrowing Behavior of Soldier Crab Mictyris longicarpus (Latreille, 1806) on North Stradbroke Island, Queensland, Australia Christopher J. Law

The soldier crab Mictyris longicarpus (Latreille, 1806) exhibited an apparent response to the ebb and flow of 12.4-hour tidal cycle, emerging at low tide and burrowing at high tide. This behavior could be influenced by environmental variables (exogenous), biological rhythms (endogenous), or a combination of both. It was hypothesized that soldier crabs exhibit an endogenous circatidal rhythm that influences its surfacing and burrowing behavior. A tidal simulated habitat and a non-tidal simulated habitat were created to determine if an endogenous circatidal rhythm solely governed the behavior or if exogenous factors, such as the tides, influenced the crabs’ burrowing behavior. The results of this study demonstrated that, within the 5-day study period, the crabs were no longer synchronized with the ebb and flow of the 12.4-hour tidal cycle. Subsequently, supplementary variables were believed to have an influence in the crab’s behavioral mechanism, with one of the most likely variables being weather. Nevertheless, further studies are required to confirm the influence of weather conditions and to investigate additional variables. Key Words: Circatidal rhythm • soldier crab • tidal cycle • circadian rhythm

Introduction

The intertidal zone is a hostile environment where the ebb and ﬂow of the tides expose organisms to highly variable factors such as temperature, sun exposure, anoxic soil, and salinity. It serves as a prime example of where organisms have evolved mechanisms to time their activities to the temporal structure of the environment, also known as the tidal cycle. Subsequently, organisms have evolved and adapted behavioral mechanisms that allow them to gain advantage of this temporal structure.1 There are three approaches the mechanism can rely on: (1) a direct stimulus response from the environment (exogenous); (2) an internal clock — i.e. circadian rhythm (endogenous); and (3) a combination of environmental and innate factors.1,2 Circadian rhythm is thought to be one of the most important behavioral mechanisms. According to Turek and Takahashi, circadian rhythm is a biological clock that allows organisms to predict and prepare for the day and night changes of an environment and drives organisms to behaviorally and physiologically adapt to meet those changes and thus form a temporal synchronization between the organisms and their environment.3 Circatidal rhythm, a type of circadian rhythm with a period length of one 12.4 hour tidal cycle which consists of an ebb

tide (low tide) and a flood tide (high tide), has been found in nearly all groups of marine organisms.2,3 Subsequently, multiple studies have shown that many intertidal organisms have behavioral mechanisms, such as the swimming behavior of the isopod Excirolana chiltoni, ovigerous blue crabs Callinectes sapidus, and mole crab Emerita talpoida, that are derived endogenously through a circatidal rhythm.4,5,6,7 This mechanism is responsible for the behaviors exhibited by each of these organisms, enabling each organism to flourish in the tidal fluctuations that characterize intertidal habitats. However, additional studies have shown that other intertidal organisms — such as the fiddler crab Uca spp. and the blenny Lipophrys pholis — have additional factors that influence their behavior.2,8,9,10,11 The soldier crab, Mictyris longicarpus (Latreille, 1806), exhibits an apparent response to the tide level. During low tide, thousands of soldier crabs form “armies” that trek across the extensive intertidal sandy shores of many southeast Queensland coastlines, grazing upon the meiofauna and detritus found within the sand (Figure 1). This behavior plays an ecologically significant role in local bioturbation with its ability to mix and “cleanse” sand by removing approximately 95% of carbon and 99% of nitrogen from surface sediments.12,13,14,15,16 However, minutes, and even hours before

Figure 1. Soldier crab off Dunwich.

tidal inundation, the crabs stop feeding on the surface and bury themselves in the sand. Consequently, a synchronicity exists between soldier crabs and the tidal cycles became known. However, the mechanism behind the soldier crab’s burrowing behavior has yet to be explored. The aim of this study is to determine whether the mechanism controlling circatidal behavior is governed exogenously through environmental factors, endogenously through a circatidal rhythm, or some combination of both. This study hypothesizes that the soldier crab Mictyris longicarpus exhibits an endogenous circatidal rhythm that controls the crab’s behavioral response to the tidal cycle, and therefore crabs exposed to tides and those not exposed to tides are expected to exhibit similar surfacing and burrowing behaviors with respect to the tides.

Materials and Methods Table 1. Number of individuals in each artificial habitat. sq.ucsd.edu

Background and setup of the artiﬁcial habitat VOL 9 • SALTMAN QUARTERLY

RESEARCH The soldier crabs (Mictyris longicarpus) used in this study originated from the intertidal sandy shores of Dunwich, North Stradbroke Island, Queensland, Australia (27°30′S 153°24′E), a location that exhibits semidiurnal tides. The entire study took place during the peak of a neap tide event, lasting from September 29 to October 3, 2010. On September 29, a total of 103 soldier crabs (Mictyris longicarpus) were collected from the intertidal sandy shores of Dunwich during the morning low tide (6:37; 0.5 m) and randomly divided and placed into either a habitat with tidal simulation (47 individuals) or a habitat with non-tidal simulation (48 individuals). Each habitat consisted of a bucket filled with enough moist sand that enables the crabs to burrow. The habitats were placed in a semi-enclosed room and had access to temperature, humidity, and pressure changes but no sunlight. The habitat under tidal simulation was subjected to artificial tidal changes based on the actual tidal changes. To simulate an artificial high tide, the habitat was submerged with enough water to cover the surface of the sand by at least 5 cm. Conversely, water was siphoned out at mean tide level to simulate the artificial low tide. The habitat under nontidal simulation was subjected to neither artificial nor natural tidal changes and was left undisturbed with no water cover. The Experiment The experiment was conducted over a period of four days, from September 29 to October 2 of 2010, and consisted of seven low tides. Both habitats were observed every 15 minutes for the majority of each day, with an increase to every 10 minutes approximately one hour before and after the low tide. To determine if a relationship between tidal height and soldier crab activity existed, three components were noted at every observation: (1) the number of crabs that surfaced from their burrows in each habitat; (2) the time of observation; and (3) the tidal height at Dunwich during the observation. In addition, brief field observations were made about whether the natural populations of soldier crabs had surfaced during low tide. A total of 68 additional soldier crabs were collected on the last day (October 2) in order to draw comparisons between the artificially induced crabs and the freshly caught ones. The new crabs were subjected to non-tidal simulation (NNT). As with the previous two habitats, the two new habitats were monitored every 15 minutes, increased to every 10 minutes during low tides, to observe the number of crabs that surfaced throughout the day. The study was ended on October 3, at which time the crabs were dug out of the artificial habitats, counted, and released back to intertidal zone of Dunwich. A total of nine T crabs, one NT crab, and one NNT crab were accounted for as dead. Data Analysis

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(A)

(B)

(C) Figure 2A-E. Surfaced crab activity throughout the 5-day study. Crab surfacing tended to take place during the low tide events. sq.ucsd.edu

RESEARCH Data was recorded from September 29 to October 3 for a total of five days. The observed peak percentages of surfaced crabs and tidal height were plotted against time. In addition, the difference between time of actual crab surfacing and time of predicted crab surfacing was calculated and termed as lag. Actual time was defined as the time at which peak surfacing of crabs was observed, whereas predicted time was defined as the time of low tide, the time at which peak surfacing of crabs was expected. The crabs that died during the experiment were included in the calculations of the percentage of crabs above ground and in the final results as there was no way of knowing when each individual had died. Statistical Analysis To determine whether each succeeding tide simulation affected the peak percentage of surfaced crab and the lag between actual and predicted times, two linear regressions were performed, respectively. Two student’s t-tests were also conducted to compare the difference in: (1) the percentage of crabs that emerged during actual low tides between the two simulations and (2) the peak percentage of crabs that surfaced using MATLAB. All data was presented as (mean ± standard error).

(D)

Results

The number of crabs that surfaced under both tidal simulation (x = 29.6% ± 12.4) and no tidal simulation (x = 26.7% ± 12.9) was highly variable between and within each day of the study (Fig. 2). Whereas morning low tides (3 occurrences) were characterized by low peak percentage of surfaced crabs (x = 8.9% ± 4.6 and x = 2.1% ± 1.2 for T and NT crabs, respectively), evening low tides (4 occurrences) were met with great variability in the peak percentage of surfaced crabs (x = 45.1% ± 18.4 and x = 38.5% ± 16.6 for T and NT crabs, respectively). The evening tides during days 1 and 3 were met with similar peak percentages of surfaced T and NT crabs (93.8% vs. 83.0% for day 1 and 26.7% vs. 18.8% for day 3). However, more NT crabs surfaced compared to T crabs on day 2 (8.9% T crabs vs. 43.8% NT crabs), whereas more T crabs surfaced compared to NT crabs on day 4 (51.1% T crabs vs. 8.3% NT crabs). The combined peak percentage of surfaced T and NT crabs during the morning low tides (x = 5.5% ± 2.6) was statistically different (Student’s t test; P < 0.05) to the combined peak percentage of surfaced T and NT crabs during the evening low tides (x = 41.8% ± 11.5). Observations of simulated habitats trend toward a decline in peak percentage of surfaced tidal simulated and nontidal simulated crabs at each subsequent low tide (Figure 3). However, regression analyses revealed that these declines in peak percentages were not statistically signiﬁcant (linear regression t test; t = 0.61; df = 10; P > 0.05) for either group of crabs. Furthermore, the peak percentage of surfaced tidal and sq.ucsd.edu

(E)

nontidal crabs (x = 29.6% ± 12.4 and x = 26.7% ± 12.9, respectively) was found to be not statistically different (Student’s t test; P > 0.05). However, there was a statistically signiﬁcant difference between lag — the actual time of peak crab surfacing and the predicted time of peak surfacing — of the two crab groups (linear regression t test; t = 2.91; df = 10; P < 0.05) (Fig. 4). The tidal simulated crabs increased in lag whereas the nontidal simulated crabs decreased in lag. Field observations Wild solider crab populations tended not to surface during the morning low tide periods but did surface during the evening low tides. Mornings of the four day study were characterized by low temperatures with cold, strong winds, whereas evenings were characterized with mild weather conditions. Rain occurred during the mornings and afternoons of day 4 and 5.

Discussion

Statistical analysis revealed that the behavior patterns of tidal and nontidal crabs were not dissimilar, probably due to the high variability in number of crabs surfacing at each subsequent low tide throughout the four-day study. This suggested that an endogenous mechanism independent of external simulations governed their burrowing behavior; however, the presence of a lag and an observed decline in peak percentage of surfacing crabs indicated that the crabs were becoming less synchronized with the tidal cycle and that additional exogenous factors may have been inﬂuencing their behavior as well. Increase in absolute lag The signiﬁcance in the difference between the lag of surfaced tidal and nontidal simulated crabs suggests that the crabs were not synchronized to an internal mechanism independent of an external simulation such as the tides. Furthermore, the rhythmic VOL 9 • SALTMAN QUARTERLY

RESEARCH pattern did not completely correspond to the ebb and flow of the 12.4-hour tidal cycle, particularly as the study progressed. The overall lag between actual surfacing time and predicted surfacing time increased for tidal simulated crabs but decreased for nontidal simulated crabs within a matter of a few days. Organisms with pure endogenous circatidal rhythms kept in artificial habitats should remain synchronized with the tidal cycle and should not exhibit drastic lag like the crabs from this study had displayed.3 Instead, these results suggests that the crabs exhibited irregular burrowing behavior by appearing earlier or later than the actual low tide, thus serving as an indication that the behavioral mechanism was not solely governed by an endogenous circatidal rhythm as originally hypothesized.2 Similar irregular circatidal rhythm have been observed in the spawning migration of blue crabs (Callinectes sapidus), in which the migrating crabs’ rhythm drifted out of phase due to external cues and therefore no longer coincided with the time of the ebb tide.6 Decline in peak percentage of surfaced crab Exogenous factors led to the decline in peak percentage of surfaced crab as well as the relatively low number of surfaced crabs during morning low tide. Overall, the peak percentage of both T and NT surfaced crabs declined every subsequential low tide during the five-day study period, and eventually the majority of the crabs from both groups did not surface at all. This suggests that the majority of the tested crabs lost their circatidal rhythm, thus demonstrating that soldier crabs were not governed purely by an endogenous circatidal rhythm. Studies on other crustaceans, such as fiddler crabs (Uca spp.), have been shown to lose their endogenous circatidal rhythm in favor of another mechanism following the day-

Figure 3. Decline in peak percentage of surfaced crabs at each succeeding tide. Linear regression of the peak percentage of crabs from every low tide period during the duration of study. Both T and NT crabs exhibit a decline in peak percentage as the study progressed. LT = low tide

night cycle, given certain environmental cues.8 Similarly, the soldier crabs may have encountered an exogenous cue that led them to stray away from their circatidal rhythm. Consequently, supplementary factors working with the circatidal rhythm must have inﬂuenced the surfacing and burrowing behavior of these soldier crabs. Weather as a possible exogenous variable The percentage of surfaced crabs during morning low tides was signiﬁcantly lower than the percentage of surfaced during evening low tides, but perhaps more

interesting was that each morning low tide coincided with overcast skies and strong, cold winds. Because the simulation habitats were kept in a semi-enclosed room, both groups were exposed to the drop in temperature as well as increase in humidity and pressure. It has been shown that cooler temperatures influence the surfacing behavior of soldier crabs.17 In addition, other studies noted that soldier crabs preferred sunny days and would seldom surface on cold, windy, overcast, or rainy days.13,15 Field observations from this study confirmed that soldier crabs indeed did not come out on colder days. It was observed that the wild populations of soldier crabs did not surface during the morning low tide periods, characterized by low temperatures with strong, cold winds. The majority of crabs in the simulated habitats also did not surface during these periods. This relationship between wild crabs and captive crabs was observed throughout the entire course of this study, especially during morning low tides; rainy and cold mornings tended to indicate few or no crab surfacings in both wild and captive crabs. The observations from Day 5 (October 3) were perhaps the most evident of this trend. The morning was characterized by rain, and subsequently, no wild crabs were observed to surface, and 100% of the T, NT, and NNT crabs remained burrowed as well. Evidently, weather, along with the circatidal rhythm, seems to influence the crabs’ surfacing behavior.

Conclusion Figure 4. Change in lag at each succeeding low tide Linear regression of the lag between actual time of surfacing and predicted time of surfacing from every low tide period during the duration of study. T crabs displayed an increase in lag whereas NT crabs displayed a general decrease in lag; however, the difference was found to be insignificant.

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Our hypothesis that soldier crabs relied solely on an endogenous circatidal rhythm was proven to be incorrect. The soldier crab, Mictyris longicarpus, demonstrated an endogenous circatidal rhythm along with sq.ucsd.edu

RESEARCH

Table 2. Lag of surfacing soldier crabs. Lag = actual time – predicted time. Acutal time was defined as the time of the peak number of surfaced crabs during the low tide period. Predicted time was defined as the time of actual low tide. In theory, crabs with an endogenous circatidal rhythm will have peak surface activity at the time of actual low tide.

supplementary exogenous factor(s) — i.e. weather — that governed its surfacing and burrowing behavioral mechanism. Data may have been skewed due to the nature of this study — there was a limited duration of study (only five days), insufficient tidal simulation equipment, unideal buckets used as habitats, and unfavorable weather; however, these limitations should not have had a tremendous impact on the results. Nevertheless, if a replication of this study were performed, four improvements should be made: (1) there should be a longer period of study with 24-hour observations, (2) additional observations of wild soldier crab populations for comparisons between the wild and captive crabs, (3) an effective tidal simulation method, and (4) a reduction of the crabs’ stress level through an increase in the size of the habitat. Further research is recommended to formally test the influence that weather has on the soldier crabs’ behavioral mechanism and to determine additional variables that may influence the mechanism. Similar studies have shown that the behavioral mechanism of many intertidal organisms is governed by multiple variables in addition to circatidal rhythms. Fiddler crabs (Uca spp.), for instance, exhibited an endogenous mechanism influenced by the tidal cycle and day-night cycle.9 Consequently, it is probable that additional variables influence the soldier crabs’ behavioral mechanism as well. Variables that future studies might consider are (1) influences of temperature and weather conditions, (2) addition of the day-night cycle, which may lead to the detection of a daily circadian rhythm, and (3) environmental factors such as salinity, physical impacts of tides, and oxygen availability underground.

Acknowledgements

We would like to thank Professor Ian Tibbetts and his teaching assistants, Natale Snape, Mitch Zischke, and Craig Chargulaf, for their advice, assistance, and support with sq.ucsd.edu

this project. Furthermore, we would like to thank the Morton Bay Research Station and its staff for the assistance and equipment needed for this study.

References

1. Horton, T.H. (2001). Conceptual issues in the ecology and evolution of circadian rhythms. Handbook of Behavioral Neurobiology. (12), 45-57. 2. Palmer, J. D. (2000). The clocks controlling the tide-associated rhythms of intertidal animals. BioEssays, 22, 32-37. 3. Turek, F.W., Takahashi, J.S. (2001). Introduction to circadian rhythms. Handbook of Behavioral Neurobiology, (12), 3-6. 4. Akiyama, T. (1995). Circatidal swimming activity rhythm in a subtidal cumacean Dimorphostylis asiatica (Crustacea). Marine Biology, 123, 251-55. 5. Forwards, R.B., Tankersley, R.A., Pochelon, P.N. (2003). Circatidal activity rhythms in ovigerous blue crabs, Callinectes sapidus: implications for ebb-tide transport during the spawning migration. Marine Biology, 142, 67-76. 6. Forward Jr., R.B., Cohen J.H. (2004). Factors affecting the circatidal rhythm in vertical swimming of ovigerous blue crabs, Callinectes sapidus, involved in the spawning migration. Journal of Experimental Marine Biology and Ecology, 299, 255-66. 7. Forward Jr., R.B., Diaz, H., Cohen J.H. (2005). The tidal rhythm in activity of the mole crab Emerita talpoida. Journal of the Marine Biological Association of the U.K., 85, 895-901. 8. Stillman, J.H., Barnwell F.H. (2004). Relationship of daily and circatidal activity rhythms of the ﬁddler crab, Uca princeps, to the harmonic structure of semidiurnal and mixed tides. Marine Biology, 144, 473-82. 9. Morgan, E., Northcott, S.J., Gibson, R.N. (1991). The effect of tidal cycles of hydrostatic pressure on the activity of Lipophrys pholis (Teleostei). Journal of Experimental Marine Biology and Ecology, 148, 35-45.

10. Faria, C. Almada, V.C. (2008). Tidal activity rhythms and depth distribution of rocky shore fish in an altered intertidal environment. Acta ethologica, 11, 123-26. 11. Northcott, S.J., Gibson, R.N., Morgan, E. (2009). The persistence and modulation of endogenous circatidal rhythmicity in Lipophrys pholis (Teleostei). Journal of the Marine Biological Association of the U.K., 70, 815-27. 12. Cameron, A.M. (1966). Some aspects of the behavior of the soldier crab, Mictyris longicarpus. Pacific Science, 20, 224-34. 13. Quinn, R.H. (1986). Experimental studies of food ingestion and assimilation of the soldier crab, Mictyris longicarpus Latreille (Decapoda, Mictyridae). Mar. Biol. Ecol., 102, 167-81. 14. Dittmann, S. (1998). Behavior and population structure of soldier crabs Mictyris longicarpus (Latreille): observations from a tidal flat in tropical North Queensland, Australia. Senckenbergiana Maritima, 28, 17784. 15. Rossi, F., Chapman, M.G. (2003). Influence of sediment on burrowing by the soldier crab Mictyris longicarpus Latreille. Journal of Experimental Marine Biology and Ecology, 289, 181-95. 16. Webb, A.P., Erye, B.D. (2004). The effect of natural populations of the burrowing and grazing soldier crab (Mictyris longicarpus) on sediment irrigation, benthic metabolism and nitrogen fluxes. Journal of Experimental Marine Biology and Ecology, 309, 1-19. 17. Kelemec, J.A. (1979). Effect of temperature on the emergence from burrows of the soldier crab, Mictyris longicarpus (Latreille). Australian Journal of Marine and Freshwater Research, 30, 463-8.

About the Author

Chris Law is an Environmental Systems Ecology, Behavior, Evolution major from John Muir College. He is graduating in 2012.

VOL 9 • SALTMAN QUARTERLY

RESEARCH

Spectrogram Correlation Detector Optimization for the North Paciﬁc Blue Whale (Balaenoptera musculus) B call Sarah Pfeil

Spectrogram correlation methods for the detection of blue whale (Balaenoptera musculus) B calls were developed and evaluated. In 2008 and 2009, long-term underwater acoustic recordings from two sites off the coast of southern California were made using high-frequency acoustic recording packages. Spectral characteristics of the blue whale B call were summarized to build a spectrogram kernel to use in the automatic detector. Optimization of the detector involved minimizing both the number of missed calls and false detections. The optimal detection thresholds differed for each site, given the difference in ambient noise conditions. False detection rates were 9% and 11%, and missed call rates were 12% and 19%. After adjusting for detector performance, the call rate was higher at the site further offshore, which is signiﬁcant because this site is farther from shipping lanes and may imply that whales avoid communication in noise polluted waters. Future studies using this method can work to reduce analysis time and ultimately help answer questions regarding blue whale abundance and distributions. Key Words: blue whales • DET curve • acoustics • automated detection • marine

Introduction

Sound is an efficient way to propagate energy through the ocean, and many marine organisms, including blue whales, have evolved complex acoustic communication systems to take advantage of this property. Acoustic monitoring of animal calls is a valuable tool for tracking spatial and temporal patterns of marine mammal species. Developing tools to effectively and efficiently analyze acoustic data for the presence of calling marine mammals remains a challenge, given the variability in call types and differences in acoustic environments. Northeastern Pacific blue whales produce three distinct call types — A calls, B calls, and D calls — that differ in acoustic frequency and duration. The first two are organized into songs as ABAB or ABBB repetitions. Song is known to be only produced by male blue whales and is thought to be associated with mating or courtship behavior.7 Still, song does occur year round and thus may have more profound social implications.9 The A and B calls that make up song are also heard as singular calls produced by males when either traveling or feeding.7 The third call type is the D call, which is a short downsweep, associated with feeding behavior and produced by both males and females.7 A blue whale B call detector developed

and evaluated in this study uses a spectrogram correlation. Spectrogram correlation is a type of time series analysis that shows how a signal changes over time and which frequency band it lies within. The result is a visual image of the sound over time characterized by the frequencies it reaches. B calls are tonal calls lasting around 10 seconds with a fundamental frequency at 16 Hz and an intense third harmonic around 48 Hz. The consistent characteristics of this call make it an ideal candidate for automated detection using spectrogram correlation. An optimization process is necessary to evaluate the performance of a detector. Speciﬁc call characteristics were used to deﬁne a kernel. The kernel is an averaged image of a typical call and takes into account frequency and duration. Signals in the acoustic data that match kernel characteristics are considered detections. However, the detector can make false detections or miss calls. For example, in the presence of lowfrequency tonal ship noise, false detections might occur. Also, if calls are faint or far away from the recording hydrophone, the detector may not be sensitive enough to pick them out from the background noise. Optimized and validated automated detection methods may greatly reduce data analysis time as well as allow entire datasets

Site Site Name

Latitude

Longitude

Depth (m)

Start Date

End Date

1 2

34-19.110 N 32-39.539 N

120-48.333 W 119-28.632 W

700 1,334

10/15/2008 6/24/2009

12/4/2008 9/6/2009

CINMS04C SOCAL34E

Table 1. Deployment locations and dates of HARP recordings. Sites 1 and 2 are both off the southern California coast, and data was recorded during the seasonal peak in blue whale occurrences.

SALTMAN QUARTERLY • VOL 9

to be examined efficiently. Current detection methods rely on manual selection by a trained acoustic analyst, although some call-specific automated detectors have been developed. The manual detection process is both timeconsuming and has biases associated with the analyst, which are difficult to quantify. Here, we develop and evaluate an automated method for detecting blue whale B-calls using a spectrogram correlation method at two sites in southern California. Acoustic recording sites were located on the feeding grounds of the northeastern Pacific blue whale population and monitored during the summer and early fall months.2 Our techniques allowed us to quantify the efficiency of a blue whale B call detector through comparisons with manual detections by an analyst. We hypothesize that the detector performance will depend on the acoustic environment of the recording site. Future studies using this method will allow for more efficient data analysis and ultimately help us answer questions regarding abundance and distributions for this endangered species.1

Materials and Methods Acoustic recordings To record blue whale vocalizations autonomous High-Frequency Acoustic Recording Packages (HARPs) were deployed at two sites off the coast of southern California (Table 1).10 Site 1 was monitored in 2008, and Site 2 was monitored in 2009. Recordings occurred during the peak in blue whale occurrence in the region, from late sq.ucsd.edu

RESEARCH # Manually Optimal % % Total DeSite Selected Threshold False Missed tections Calls 1 2

2,782 1,881

60.7 49.1

8.5 11

12.2 18.5

47,598 70,209

% Error 0.05 0.03

Total Calling Rate True Calls Es(Calls/Hr) timate 49,377 40 75,447 71

Table 2. Summary of detector performance for each site, including detector thresholds, false and miss probabilities, total detections, total true calls, and calling rate. Error rates for both detectors were minimal and the optimal threshold of the detector was higher at the noisier site, Site 1.

summer to early fall. HARPs recorded at a sampling frequency of 200 kHz and continuously sampled over the survey period. Site 1 is characterized by high levels of ambient shipping noise as it is located approximately 5 km from major commercial shipping lanes (shown in white, Fig.1). In contrast, site 2 has lower average noise levels. Manual call selection Manually selected B calls served as the ground truth for the detector performance. B calls were chosen based on the presence of the third harmonic at 48 Hz because the signal to noise ratio (SNR) is higher than the fundamental frequency (16 Hz). Approximately 3 days of data from each deployment were chosen at random, and all the B calls were manually selected using Triton Logger, a Matlab based software.6 Building the B-call kernel A synthetic kernel, or reference function, representing the B call third harmonic was created. The kernel was divided into four segments at intervals of 1.5 seconds, 1.5 seconds, 1.5 seconds, and 5.5 seconds for a total duration of 10 seconds. The start and end frequencies of each segment were averaged from 30 high-quality, hand-selected B calls from independent calling bouts that spanned the entire deployment. A separate kernel was created for each deployment using this method. A 2 Hz bandwidth was set around each kernel to allow for some call variability. Optimal detector parameters To calculate the spectrograms used with the detection algorithm, fast Fourier transforms (FFTs) of the time-series waveforms were performed with 2,048 samples, 50% overlap, and Hanning windows. Once the images of the acoustic data were created, these spectrograms were cross-correlated, or compared, with the synthetic kernel. The output of the spectrogram cross-correlation was a recognizing score function. This function scores the detection against the parameters sq.ucsd.edu

of the synthetic kernel. When the function score was greater (matched more) than a user-deﬁned threshold for a given duration, the detection of a call was noted, and the start time was recorded. If the detection threshold was set too low, then many false detections result; low detection thresholds are more sensitive, detecting sounds that are not necessarily B calls, thus resulting in many false detections. On the other hand, if the detection threshold was set too high, then many calls were missed. High detection thresholds are very selective and therefore only detect very clear, loud, characteristic B calls that match the shape, frequency, and duration of the deﬁned kernel closely. Thus, high detection thresholds result in many missed calls. In order to get an optimal threshold value, automated detection results were collected with the threshold set low enough to ensure that the miss rate was 0. These results were compared to the results of manually selected calls. The probability of false detections versus the probability of missed calls was plotted in a standard Detection Error Tradeoff (DET) curve.3 A 5:1 missed to false detection cost ratio was set, and optimal thresholds for the two datasets were calculated. The 5:1 ratio was chosen so there is a low false alarm probability while keeping the missed calls to a minimum. Detection

results were then obtained from each dataset at the corresponding optimal threshold. Evaluating the detector The false and missed probabilities were used to adjust the total number of B call detections from each dataset. Detection results were veriﬁed against the subset of manually selected calls corresponding to each dataset. This allowed an evaluation of detector performance using percent error calculation: [Ddt + ((Ddt * missed) - (Ddt * false))] - Dmt Dmt

*100

Where Ddt is the number of detections in time t, Dmt is the number of detections manually selected in the same time period, false is the probability a of false detection, and missed is the probability of missing a call.

Results

B call characteristics The B call kernels used in the spectrogram correlation had an average of 30 or more independent calls from the entire deployment. The kernels were designed to be made of four segments to accommodate the beginning of the down-sweep in the tonal call. A comparison of the frequencies associated with the down-sweep of the third harmonic resulted in no difference related to site or year, as evidenced by the huge standard deviations for each kernel (Fig.2). The shape of the kernel at Site 2 in 2009 differed slightly from the shape of the kernel for Site 1; however, the shape difference was in the beginning segments of the down-sweep, and this portion is the most variable segment of the call. The differences were not deemed signiﬁcant. The similarity in kernel shapes across sites and consecutive years suggests that the same kernel can be

Figure 1. Map of deployment sites and relation to shipping lanes. Site 1 is approximately 5 km from the commercial shipping lanes shown in white. VOL 9 • SALTMAN QUARTERLY

RESEARCH Discussion

Figure 2. Spectrogram correlation kernel comparison. Error bars are ± standard deviation of the calls used to find the averaged kernel. While some difference is seen in the down-sweep, it is not statistically significant.

used to detect calls at different sites. Detector performance The detector performance was summarized for each deployment using standard DET curves (Figs.3 and 4). The thresholds listed on each detection curve and in Table 2 were the thresholds given for the deployments using a 5:1 call cost ratio of missed calls to false detections. These detection curves reflect revised ground truth picks after the first detector trials in each dataset. The curves are such that different percent miss and false results can be achieved in different study situations by adjusting the threshold. Thus, different situations may use different optimal thresholds for the detector. The optimal detection thresholds differed for each site, given the difference in ambient noise conditions, as hypothesized. In order to maintain a 5:1 call cost ratio, false detection rates were 9%, and missed call rates were 12% at Site 1, and 11% and 19% at Site 2 (Table 2). Estimates of total calling and call rate The estimated total calls based on the detector performance for each deployment are listed in Table 2. The detections were adjusted based on the known detector error (percent false and percent missed). These estimated numbers of total true calls were compared to the ground truth numbers for a given file, and the percent error was calculated. As expected, the error rate was

SALTMAN QUARTERLY • VOL 9

low (Table 2). Deployment 1 recorded data for 48 days, and Deployment 2 recorded data for 41 days. Even though Deployment 2 was shorter, more true calls were detected. This recording period occurred earlier in the blue whale season and at the offshore site (Site 2). As expected, calling rate per hour was also higher at this site (Table 2).

Spectrogram correlation proved to be an adaptable and useful detection method for blue whale B calls. The DET curves were useful for finding and adjusting the threshold of the detector to set a specific cost ratio and can be used in future studies with different detection objectives. The optimized detector using a manually defined kernel, 5:1 missed to false call cost ratio, and the optimal threshold for each dataset yielded extremely low rates of error at — less than 1% at each site. The low error rate in both datasets and the similar kernel characteristic implies that the same kernel may be used for automated detection in datasets from either site in consecutive years. The main differences observed between deployment sites were the slight frequency differences at the beginning of the downsweep and the different optimal thresholds needed to accurately run the detector through the spectrograms. The difference in the beginning down-sweep of the B call was not significant from 2008 to 2009 across sites in this study. While no significant tonal down shift was seen from 2008 to 2009, this is still highly useful as it allows for the same synthetic kernel to be used in consecutive years at these two sites, which ultimately reduces analysis time. The optimal thresholds differed between sites. This is likely related to the ambient noise level, which was much lower at Site

Figure 3. Detection curve for Site 1(CINMS04C) showing percent false and missed detections as well as the optimal threshold using a 5:1 call to cost ratio (missed to false ratio). The optimal threshold is encircled. A user defined threshold of 60.7 will yield a total number of detections, 8.5% of which will be false and 12.2% of the total true call amount will be missed. N=10,581 where N is the total number of detections at threshold 25. sq.ucsd.edu

RESEARCH 2 compared to Site 1. The low-frequency noise levels at 40 Hz, which is within the B call range, for Site 1 was 88 dB re 1 µPa2/ Hz on average; this was much higher than the average of 80 dB re 1 µPa2/Hz measured at Site 2. The difference in average noise levels between these two sites is likely related to the proximity of shipping lanes to Site 1 (Fig.1).8 Not only does the average noise level at each site affect the optimal threshold by demanding a higher threshold in noisier environments, but it may also impact blue whale calling behavior. Site 2, the quieter site farther away from the shipping lanes, showed a calling rate of 71 calls per hour; almost double that of Site 1, which showed 40 calls per hour.5 However, the differences in calling rates are more likely related to differences in year and recording season. Overall, an increase in the total number of calls and calling rate per hour can be seen over time; however, the recording sites and exact time periods analyzed differed between the years. Therefore, we can only speculate about the reasons for this increase. For example, the increase might be related to a temporal increase in population size or a spatial difference in prey distribution. While the difference between the total calls studied from 2008 to 2009 was quite large, a population increase from 2008 to 2009 is unlikely due to the generation time and age of sexual maturity for the species. The greater number of calls and higher calling rate in

2009 at the offshore site may be related to increased calling during the beginning of the season. The detector performance surpassed what was expected and, when optimized, showed very low error rates. Also, the fact that the kernels from the two sites were so similar means that one kernel may be used in multiple datasets. This means that individual site and deployment speciﬁc kernels are not necessary for call detection at these sites during consecutive years. This further implies that the downward tonal shift in B calls over time may not necessarily be seen on a short one year time scale. However, this does not negate the observed downward trend in B call tonal frequencies over larger time sclaes. Given the observed frequency decrease in blue whale B calls, the spectrogram correlation kernel should be evaluated every few years.4

Conclusion

The optimized detector developed and tested in this study proved to be highly efﬁcient for both acoustic datasets analyzed. This accuracy, combined with the similarity in spectrogram kernel characteristics, allows for future analysis at different sites using the optimized detector. This would signiﬁcantly improve analysis time and with further optimization my increase accuracy beyond traditional methods. While the same kernel may be used for B call detection in different datasets, the optimal threshold will still need

Figure 4. Detection curve for Site 2 (SOCAL34E) showing percent false and missed detections as well as the optimal threshold using a 5:1 call to cost ratio. The optimal threshold is encircled. A user defined threshold of 60.7 will yield a total number of detections, 11% of which will be false and 18.5% of the total true call amount will be missed. N=6,512, where N is the total number of detections at threshold 25. sq.ucsd.edu

to be evaluated in order to obtain the same low error rates. Lastly, the difference in calling rates observed at sites 1 and 2 in 2008 and 2009 may be due to differences in seasonality of migration patterns, average noise levels, or other behavioral changes in the population. Future investigations of calling rate with diel patterns as well as seasonal patterns may reveal more information about blue whale ecology and behavior in the region.

Acknowledgements

The lead author would like to thank John Hildebrand and everyone else in the Scripps Whale Acoustics lab at Scripps Institution of Oceanography for a wonderful working and learning experience. Acoustic data were made available with the help of Sean Wiggins, Chris Garsha, Brent Hurley, and Hannah Bassett. Dave Mellinger developed the Ishmael based MATLAB code. Lisa Munger developed the Logger for Triton. Daniel Arias, Simone Baumann-Pickering, and Marie Roch offered suggestions and guidance on the project and helped develop MATLAB code.

References

1. Calambokidis, J. et al. (1990). Sightings and movements of blue whales off central California 1986-88 from photo-identification of individuals. Rep. Int. Whal. Comm, 12, 343-347. 2. Fiedler, P.C. et al. (1998). Blue whale habitat and prey in the California Channel Islands. Deep Sea Research Part II: Topical Studies in Oceanography, 45, 1781-1801. 3. Martin, A.F., G. Doddington, T. Kamm, M. Ordowski, M. Przybocki. (1997). The DET curve in assessment of detection task performance. Proc. Eurospeech ‘97, 4, 1899-1903. 4. McDonald, M, J.A. Hildebrand, S. Mesnick.(2009). Worldwide decline in tonal frequencies of blue whale songs. Endangered Species Research, 9, 13-21. 5. McKenna, M.F. (2011). PhD Thesis, UCSD Scripps Institution of Oceanography. June 2011. 6. NOAA Fisheries Office of Protected Resources. (2011, January 6). Blue Whale (Balaenoptera musculus). Retrieved from http://www.nmfs.noaa. gov/pr/species/mammals/cetaceans/bluewhale. htm 7. Oleson, E.M. et al. (2007). Behavioral context of call production by eastern North Pacific blue whales. Marine Ecology Progress Series, 330, 269–284. 8. Ross, D. (2005). Ship sources of ambient noise. IEEE Journal of Oceanic Engineering, 30, 2. 9. Stafford, K., Nieukirk, S.L., Fox, C.G. (2001). Geographic and seasonal variation of blue whale calls in the north Pacific. Journal of Cetacean Research and Management, 3, 65–76. 10. Wiggins, S.M. J.A. Hildebrand. (2007). Highfrequency Acoustic Recording Package (HARP) for broad-band, long-term marine mammal monitoring. International Symposium on Underwater Technology 2007 and International Workshop on Scientific Use of Submarine Cables & Related Technologies (Tokyo, Japan).

About the Author

Sarah Pfeil is a General Biology major from Earl Warren College. She graduated in 2011.

VOL 9 • SALTMAN QUARTERLY

RESEARCH

Gender affects behavior, variation in size, and site fidelity in fireflies (Coleoptera: Lampyridae Photinus sp.) Kim N. Vu

Fireflies are renowned for their bioluminescence, but have otherwise been understudied by the scientific world. This is evident by the disproportional lack of studies done on their behavior in comparison to the amount of research devoted to the molecular biochemistry of their light. In this study, I captured and marked 98 fireflies in one pasture over the course of two weeks, and noted recaptures and gender based on behavior. I found that male and female fireflies of Photinus sp. exhibit distinguishable courtship and movement behavior from which their sex can be identified. Otherwise, it is very difficult to distinguish between the two. Light signaling in flight typify males, while light signaling from the ground typify females. Frequent flight is characteristic of males, while females spend a majority of their time crawling or remaining stationary. Females are also slightly larger than males in this species. The absence of recaptures after 24 hours of both males and females indicate a lack of site fidelity. Same night recaptures suggest that fireflies remain within a smaller area per night. Key Words: lampyridae • territoriality • Costa Rica • size • recatch • site tenacity

Introduction Fireflies are soft beetles of the family Coleoptera-Lampyridae and emit bioluminescence from their abdomens to search for mates during mate selection. This is the most efficient light production currently known; nearly 100 percent of the energy used is converted into light. In comparison, an incandescent lightbulb emits only 3 percent of its energy as light while the remaining 97 percent is lost as heat.1 The production of firefly light is fueled by the enzyme Luciferase, unique to fireflies, and is of particular importance; its wide application ranges from extensive use in molecular biology to cancer research to incorporation in making space shuttles.2 However, fireflies have been underrepresented in scientific research as organisms rather than Luciferasecarrying vessels. Compare for example a search on Biosis for “Lampyridae,” which turns up 442 results, in contrast to “Luciferase,” which turns up 31,560 articles. My study seeks to study the light of the firefly as it pertains to the species from which it comes. In this study, I explore the significance behind firefly light signals and how fireflies may use it to distinguish gender during mate selection. Fireflies signal that they are ready to

Figure 1. The species of interest in this study, Photinus, sp., from the anterior view. I found this firefly more than any other along the streets of Monteverde.

reproduce by ﬂicking their lights in highly speciﬁc patterns. Males generally court females through aerial light displays, while

•

Flash in ﬂight

Males •

Females Flash from the ground

•

Are often in ﬂight

•

Are often sedentary and grounded

•

Once handled, attempt escape through ﬂight

•

Once handled, crawl faster and refrain from using wings

•

Use wings for long distances (meters, often out of eyesight)

•

Use wings for short distances (usually only a few cm at a time)

•

Use wings for larger durations of time (a few minutes)

•

Use wings for short increments of time (a few seconds)

Table 1. Males and females exhibit distinctly differential behavior.

SALTMAN QUARTERLY • VOL 9

females respond with light signals from the ground.3 Although females of most species possess the ability to fly, they stay grounded for the majority of the courting process while their male counterparts partake in the flying and searching.4 However, aside from this behavior, it is difficult to identify the gender of fireflies based solely on appearance. For some fireflies, the sex determining genitalia are internalized, as is the case for the species of firefly that I investigated in this study. I also found no perceivable sexual dimorphisms. Since it has been documented in some species that male fireflies will flash only in flight while females will flash only from the ground, I decided to investigate if this behavior was exhibited in the species. sq.ucsd.edu

RESEARCH

Figure 2. The species of interest in this study, Photinus, sp., from the lateral view.

Assuming that males spent more of their time in ﬂight than females, I also investigated the home range of males versus females and the site loyalty for both genders. I equated site loyalty as a possible indication of territorial behavior and examined larger size as a possible factor for more territorial individuals.

Materials and Methods

I made all observations and collected all data within the two-week period between 11 May 2011 and 24 May 2011. The study site encompassed a 100x42m pasture and the surrounding hedges and paths (an additional radius of about 50m) in the neighborhood of Bajo del Tigre in the town of Monteverde, Costa Rica. I collected data between 1745h and 2330h for six consecutive days and an additional four days. I could clearly identify this species from the other 2 firefly species inhabiting the area because of their blue colored, short, repetitive light flashes as well as physical features such as the patterns on their heads and elytra. The sizes of their heads and bodies were also distinguishable from other species in the field. I caught fireflies by searching for firefly flashes, then approaching individuals and capturing them by net if they were in flight, or by hand if they were grounded. Determining Gender Through Dissection I caught four fireflies that were flashing during flight and five fireflies that were flashing from the ground on three different days. I marked them, combined them, and stored them in alcohol before dissection. I did blind dissections on the abdomen of each individual without referring back to sq.ucsd.edu

whether I had caught it while it was flying or while it was on the ground, so that I could determine gender with certainty (the genitalia were extremely small in size, and overlooking the 2mm aedeagus genitalia in a male would have incorrectly indicated female). For each firefly, I determined gender by the presence or lack of presence of the male genitalia aedeagus structure in the posterior end of the abdomen (Fig. 3a), and identified fireflies as female if there were distinguishable egg-containing ovaries. Females are also more likely to have a higher abundance of globular fat stores in their abdomen region than males (Fig. 3b). Determining Movement and Territoriality Through Recaptures I caught 98 fireflies that were flashing and marked them on the elytra with acrylic

paint pens such that each individual throughout the study had a unique code. I also measured the length of the elytra to two significant figures using a digital caliper, a common method of measuring body size in firefly research.6 I handled fireflies very minimally, only to mark and measure, before returning them to their original spots and flagging the positions. I also noted the time of capture for each firefly. If I re-caught any fireflies, indicated by previous paint markings, I noted the time, flagged their new location, and measured the distance between the new location and its original sighting. I considered recaptured individuals and those with longer site tenacity to be exhibiting preliminary territorial behavior; I analyzed larger size as a possible factor for increased territoriality by comparing the average size of recaptured individuals with the average size of regular catches. I also compared the averages using a Mann Whitney U Test since the data for the recaptured individuals were not normally distributed. Evaluating Mark Resilience I captured and marked three fireflies in the same manner as I marked every other firefly in this study. I ran faucet water over them for two minutes each and examined the integrity of the marks. Marks remained fully conserved after submersion under a flowing sink faucet. Immersion of specimens in alcohol for two weeks also revealed no fading of marks from the elytra.

Figure 3. Firefly genitalia are distinctive between sexes. Determination of gender was confirmed by dissections and locating of the sex organs. The internal reproductive organs of fireflies include (a) males’ aedeagus structure in the posterior end of the abdomen (b) Egg-containing ovaries of the female across the middle of the abdomen. VOL 9 • SALTMAN QUARTERLY

RESEARCH or female. Size Variation A normal bell curve distribution is present in both genders. My sample population included elytra length ranging from 9.50 mm to 12.75 mm ( = 11.08mm, σ = 0.55 mm). My sample population is disproportionally female (76 females), which is more than 3.5 times that of my male population size (21 males). The average length of elytra was significantly larger in females ( =11.14mm, σ = 0.56 mm) than in males ( = 10.84mm, σ = 0.47mm) (Mann-Whitney U test, Z=-2.34, p=0.019, Figure 4). Recaptures and Movements I had 11 re-captures out of 95 total firefly catches (11.6 percent re-capture rate). However, these were all same night recaptures and there were zero re-catches of fireflies marked from previous nights. I recorded one individual female’s movements over the duration of 42 minutes to gain insight on female movement. Initially, the specimen was flashing light signals, but did not do so during the observation period after being caught, marked, and released. Territoriality I found no evidence that the average size of re-captured females was larger than the population average (Mann-Whitney U Test, Z=1.12, p=0.26).

Discussion

Figure 4. (a) Size distribution of total samples based on elytra length. A normal distribution indicates a normal sample population in terms of size. (b) Size distribution by gender based on elytra length. The female population accounts for a higher proportion of the variance and study population, more than tripling the population of males (n=76 females vs. n=21 males). Both genders exhibit a bell shaped distribution.

Assessing Caliper Precision I captured and marked five fireflies, and measured the elytra of each firefly using the same digital caliper that I used to measure every firefly throughout this study. I recombined the fireflies and measured the elytra of each individual again without referring back to its previous measurement. I measured each firefly a total of five times, then averaged the standard deviations obtained from all five fireflies to determine the precision of measurements using the caliper. The average standard deviation of my measurements was 0.10mm.

Results

Gender Determination I accurately determined gender by behavior through dissections with a rate of 100 percent accuracy (n=9, 4 male and 5 female). My predictions were based on the fireflies’ location at the time of flashing in either (1) aerial or (2) grounded positions. All fireflies caught while flashing in the air

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were determined to be male, and all fireflies caught while flashing on the ground were identified as female through dissection and identification of genitalia. During my catches, I observed that fireflies exhibited distinctly differential reactions to handling based on their location of capture from either the air or the ground (See Table 1). Fireflies that were caught while signaling in the air had a tendency to take flight as soon as they were being handled. If I prevented their escape, they made continual and repetitive attempts to escape through flight. On the other hand, fireflies caught while signaling from the ground rarely utilized their wings and exhibited crawling behavior once handled. Attempts at escape through flight were rare, and the majority of them restrained completely from flexing their wings. Through dissection and differential behavior during handling, I established a criterion to identify gender in this species; I shall hereby refer to the two as either male

I found that I could identify the gender of fireflies of Photinus sp. based on movement behavior and location of signaling. Elytra length measurements revealed that females are on average larger than males in this species. The re-catch rate implies that while a nightly home range could exist, there may be no overnight site fidelity. Gender Determination Since all physical dimorphisms between male and female fireflies are internalized, the ability to determine gender without dissection is critical for behavioral and field studies. As observed in other species of Photinus sp, I found that I could distinguish firefly gender in this species based on location of flash signaling; fireflies that were flashing in flight were strictly male, 7 while fireflies that were flashing from the ground were female. Previous evidence indicates that patterns of flashing are sufficient for males and females to distinguish sexes. This study reveals that signal location may also play a role. It may be interesting to examine if signaling location alone is sufficient. A possible explanation for the development of gender based signaling location is the increased efficiency of the species in allowing female fireflies to remain near strategic egg laying sites until males fly to find them. Even though males do crawl on the ground and females do take to flight, I never observed this in conjunction with flashing. The gender-specific movement behavior I observed during firefly mate search is useful sq.ucsd.edu

RESEARCH since I observed handled fireflies returning to their locations and often resuming normal flashing activity. In this study, territorial individuals were not found to be larger. However, since I only had 11 re-captures as a sample to evaluate possible territorial behavior, additional studies where firefly capture rate is not so limited should be done to confirm these results.

Figure 5. Average elytra length comparison of male and females. Females are significantly larger.

since fireflies are not always bioluminescing. Females do not engage in flight nearly as often as males, a behavior which may persist outside the realm of courting, although it has been previously documented only as a mating behavior. There appear to be no differences in elytra functionality between males and females. Size Variation The normal bell curved distribution is expected and affirms that my capturing methods did not result in a skewed sample population, and that there is normal variation at the very least in terms of size. I found that females in my population have longer elytra than males, which indicates larger bodies. This has been documented in other species of fireflies and is common in the insect and beetle world.9 Physiologically, female firefly anatomy dictates a need for a larger volume to contain essential female reproductive organs, such as ovaries and eggs. Females’ abdomen regions also have a higher amount of fat content. Site Tenacity, Movement, and Territoriality My complete lack of re-catches from previous nights shows that fireflies probably had no overnight site fidelity to my study area. Since the capturing period spanned 10 days, six of which were consecutive, and the life span of a firefly is about two weeks, the fireflies probably never returned to my site during their lives.10 Fireflies could have been moving to new sites every night that were at least 100 meters away from the site of previous nights. The exact distance of firefly movement between nights and population movement is of great potential for future studies. Same night recaptures present insight on the movement of females in contrast to that of males. Females are much more sedentary in comparison to males. The idle behavior of females is confirmed by my case study of one female over an observation period of 42 minutes, during which she moved slowly or not at all. Based on these results and sq.ucsd.edu

the male aerial role during mate selection, I suggest that the males of this species are dependent on the distribution of females. An explanation for lack of recaptures is increased predation of fireflies due to a more conspicuous appearance after being marked by colorful acrylic paint pens. However, there were few animals in the area that would feed on fireflies.11,12 One predator that I did observe were spiders, but spiders leave exoskeletons of their victims; I never encountered any painted firefly carcasses during daytime visits to the site. The lack of painted firefly bodies also renders the explanation of acrylic paint having lethal affects on the fireflies unlikely. Additionally, I witnessed three live, marked specimens with no apparent detrimental effects from the paint. They survived for more than 24 hours before heading to the dissection board. Finally, it is unlikely that handling the fireflies scared them away from returning to the site,

Limiting Factors All re-captures and catches were limited by two things. For one, I could only catch fireflies that I sighted, which was contingent upon the fireflies’ bioluminescent patterns. Since it was nearly impossible for me to find an unlit firefly in the dark, my study only includes fireflies that were attempting to reproduce. Secondly, I could not catch every flashing firefly. Flying males includes fireflies that were attempting to reproduce.

Conclusion

The fireflies belong to a family (Lampyridae) composed of extreme behavioral diversity, such that current research has not reached the scope of many Central American species like the Photinus sp. Although Central America contains the most species of fireflies, much of the focus has been placed on the fireflies of North America, the UK, and Eurasia. Furthermore, little is known about fireflies in their natural habitat, as the majority of firefly studies are conducted in the laboratory under controlled conditions. Working in the field has allowed me to discover firefly behaviors that allow gender differentiation. My findings demonstrate that population movement and site fidelity in fireflies may be much more closely related to gender and the mating process than previous firefly studies indicate. Since firefly behavior

Figure 6. Proportion of catches that were recaptured later in the night. Recaptured fireflies demonstrated loyalty to the site for the night. VOL 9 • SALTMAN QUARTERLY

RESEARCH firefly studies indicate. Since firefly behavior is so variable among species, my findings are similar to previous research within the genus Photinus,9 but completely contrary to other work about other members of the family outside the genus.10 Although fireflies are popular and well known throughout the world, there still remains much to be elucidated about them aside from how their light organs function. There is much room for future studies on location based signaling in gender determination and territoriality, home range distances, and overnight ranging between sites. Fireflies are so commonly known, yet so little is known about them behaviorally; their beauty alone warrants additional research on other aspects of these organisms and their behavior.

Figure 7. Time and distances between initial capture and recapture of fireflies. All 10 recaptured females were found less than one meter away from their original location, ranging from five to 57 minutes after initial capture ￣= 0.8m + 1.5m over ￣ (X X =20 minutes). The single male recapture was found 64 meters away after 15 minutes.

Acknowledgements

Muchas gracias to my magical helpers Liam Soffer, Andrés Chinchilla, and Martín Campos. Marvin So, you helped me much more than you know. It’s really not that easy to stay sane in the dark catching fireflies every night at 11 without someone as marvelous as you to talk to! I’m very appreciative to Justin Penn, Chris Lee, and Dr. Henter for putting up with my tornado-like drafts. Patricia ‘Pati’ Ortiz saved this project from hopelessness many times, being the incredible advisor she is, but she is also a phenomenal artist, whose song “Fireflies” may have actually inspired all of this originally. Go watch it on Youtube please (search noctiluz video musical). Frank Joyce is awesome in many ways, but I’m pretty sure he already knows that. Thanks Frank! Additional advising

and entertainment provided by Sofía ‘gossip queen’ Arce Flores and Federico ‘tede’ Chinchilla. My heart goes out to the Trostles y Sofía y Justin for letting me ﬂag, run around in, stargaze, and take naps in their beautiful pastures.

References

1. Cary, A. (2002). Light in flight: Understanding the magic of fireflies. GORP. Orbitz Away LLC. Retrieved from http://www.gorp.com/weekend-guide/ travel-ta-light-in-flight-sidwcmdev_053049. html 2. Gayton, M. (2002). Lampyris noctiluca (European Glow-Worm). Regents of the University of Michigan. Retrieved from http://www.conknet.com/~planter/ garden/fireflies.html 3. Branham M.A., Greenfield, M.D. (1996). Flashing males win mate success. Nature, 381, 745–746. 4. Marlin, B. (2011). Firefly, North American insects and spiders. Tree Encyclopedia. Red Planet Inc. Retrieved from http://www. cirrusimage.com/ beetle_firefly_Photuris_ lucicrescens.htm 5. Caballero, S.Z. Cantharoidea de mexico nuevas species de photinus (Coleoptera: Lampyridae: photinini). Figure 8. Movement of one female over time. The female remained Ser. Zool., 67(1), 123-149. sedentary for about two thirds of the 42 minutes of observance. In 6. Vencle, F.V., Carlson, the first interval of movement, she crawled five cm in five minutes, A.D. (1998). Proximate

mechanisms of sexual selection in the firefly Photinus pyralist (Coleoptera: Lampyridae). Journal of Insect Behavior, 11(2), 191-207. 7. Cratsley, C.K., Lewis, S.M. (2003). Female preference for male courtship flashes in Photinus ignites fireflies. Behavioral Ecology, 14(1), 135-140. 8. Stanger-Hall, K.F., Lloyd, J.E., Hillis, D.M. (2007). Phylogeny of North American fireflies (Coleoptera: Lampyridae) implications for the evolution of light signals. Molecular Phylogenetic Evolution, 45(1), 33-49. 9. Kaufman, T. (1965). Ecological and biological studies on Ilie West African firefly Luciola discicollis (Coleoptera: Lampyridae). Entomological Society of America. Annals of the Entomological Society of America, 58(4), 414-426. 10. Wing, S.R. (1984). Female monogamy and male competition in Photinus collustrans (Coleoptera: Lampyridae). Psyche, 91, 153–160. 11. Buschman, L.L. (1984). Biology of the firefly Pyractomena Lucifera (Coleoptera: Lampyridae). The Florida Entomologist, 67(4), 529-542. 12. Lewis, S.M., Cratsley, C.K. (2008). Flash signal evolution, mate choice, and predation in fireflies. Ecology, 14(1), 135-140.

About the Author

Kim N. Vu is a Molecular Biology and Visual Arts- Media double major from John Muir College. She will be graduating in 2012.

and in a second interval, she crawled one cm in 10 minutes.

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sq.ucsd.edu

RESEARCH

Identiﬁcation and Characterization of Circadian Input Kinase (CikA) Localization Partners in Syenchococcus elongatus PCC 7942 Michelle Hoang and Stephen Leung

Synechococcus elongatus PCC 7942 is the simplest unicellular organism in which an endogenous biological circadian oscillator has been documented. The cyanobacterium has a circadian clock that is controlled by a central oscillator, the KaiABC protein complex. A key input protein, CikA (circadian input kinase), uses redox to sense light indirectly based on photosynthetic activity, and relays information regarding the external time to the oscillator.1 When tagged with a green fluorescent reporter gene, CikA can be seen clearly localized to the poles of the cell.5 Bioinformatics have shown that CikA contains a pseudo-receiver domain (PsR) that is predicted to function as the domain that interacts with peripheral membrane proteins to position CikA at its specific pole position.5 Mutation of CikA’s PsR domain results in mis-localization of CikA within the cell.5 In order to identify potential partners that localize CikA, a microscopic screen of single-gene knockouts encompassing the entire genome was performed, based on the hypothesis that mutants of a CikA-interacting factor will likely cause CikA to be mis-localized, similar to the pattern observed for the CikA-∆PsR mutant. Upon completion of the microscopic screen, 163 potential candidates have been found. Candidates from the genetic screen will undergo further genetic and biochemical analyses in order to confirm protein-protein interactions, and identify additional components involved in the circadian pathway. Key Words: Cyanobacteria • CikA • synechococcus elongatus • circadian rhythms • localization

Introduction

The circadian clock of S. elongatus consists of a central oscillator composed of three proteins, KaiA, KaiB, and KaiC, with input of external information from CikA. The circadian rhythm is demonstrated through an oscillating series of phosphorylation, dephosphorylation, and binding reactions within the KaiABC complex.1 KaiA interacts directly with KaiC to shift the equilibrium toward phosphorylated KaiC.1 KaiB, on the other hand, serves to negate KaiA, thereby shifting the equilibrium back toward unphosphorylated KaiC.1 CikA senses the ratio of different states of KaiC that are regulated by light and cell metabolism, and relays the redox information to the central oscillator.1 CikA is a histidine protein kinase (HPK) that serves a functional role in resetting the clock.1 When subject to a 5-hour dark pulse, the cells often reset their rhythms by up to 8 hours.1 In the absence of CikA, this 5-hour dark pulse fails to trigger a reset of the clock.1 CikA’s HPK activity is modulated by the GAF and pseudo-receiver (PsR) domains which are essential to its measurable kinase activity (Figure 1).5 The GAF domain is a positive regulator of CikA, while the PsR domain is a negative regulator.1 The PsR domain regulates CikA through proteinprotein interactions to alter the conformation of CikA.5 Research shows that once CikA reaches its proper position in the cell, the GAF and PsR domains serve as negative regulators.5 The current model suggests that the PsR domain also acts as an interaction module to dock CikA to the poles of the cell as seen in Figure 2-A. When CikA reaches its proper location at the poles through the help of its PsR domain, it is thought to form sq.ucsd.edu

Figure 1. The domains of CikA. Bioinformatics show that CikA consists of three domains essential for proper functioning: a GAF (PF01590), a histidine protein kinase (HPK) (PF06580), and a response-regulator receiver domain (PF00072).7 The GAF domain is found in phytochromes, regulatory photoreceptors which exist in 2 reversible forms that are interconvertible by light.3 S. elongatus, however, does not use photoreceptors to set the clock; instead, a key input protein, CikA (circadian input kinase), uses redox to sense light indirectly based on photosynthetic activity and relays the temporal information to the central oscillator.1 The receiver domain listed by Pfam was renamed the “pseudo-receiver” domain (PsR) in the Golden Lab because it lacks the aspartic acid residue needed to accept a phosphate from the HPK.4 The PsR domain has been predicted to interact with peripheral membrane proteins to position CikA at the cell pole.5

a large complex with other partners that are also localized to the poles.5 The aim of this study is to identify these potential partners.

Materials and Methods

The S. elongatus library of singlegene knockout mutants has been comprehensively organized into twenty-six different 96-well plates. UGS (unigene set) plates 1-9 are chloramphenicol resistant (CmR) and remaining plates 10-26 are kanamycin resistant (KmR).2 S. elongatus is a model organism when it comes to DNA transformations. Unlike other bacteria that require more complicated transformation techniques such as electroporation, S. elongatus naturally incorporates exogenous DNA into its genome. Nevertheless, transformation efﬁciency is dependent on the purity of the plasmid and growth conditions, which include proper light intensity and a temperature of approximately 30 °C. Micropipettes were used to introduce 3 µl of plasmid DNA from Escherichia coli into

each well of the 96-well plates. The E. coli plasmid contained CikA tagged with the ZsG ﬂuorescent gene, and antibiotic resistance to spectinomycin (Sp) and streptomycin (Sm).5 The plasmid was prepared from QIAGEN Mini and Maxi preps from E. coli cells grown in Luria-Bertani medium. After transforming the plasmid into the UGS mutants, the 96-well plates were individually wrapped in aluminum foil to produce darkness and placed in the 30 °C incubation room overnight. On the following day, transformants from the 96-well plate were microspotted (5 µl) onto agar plates with BG-11(N+) medium, Sp+Sm antibiotics and either Cm or Km, depending on their UGS plate of origin. Transformations were maintained on both agar plates and in 96-well plates, to allow for maximal coverage of the library because some transformants grew better on solid media while others favored liquid media (Figure 2). Each agar plate was labeled in a grid VOL 9 • SALTMAN QUARTERLY

RESEARCH format from A to H vertically and 1 to 12 horizontally, in correspondence with the original 96-well plate library. Antibiotics on the agar plate served as a selection for the S. elongatus cells that were successfully transformed with E. coli plasmid containing resistance to Sp+Sm. The microspot plates were placed at 30 °C for 3-4 days at high or medium light intensity. This incubation gave time for non-transformants to die, and positive transformants with Sp+Sm resistance to grow. Once cultures from the microspot plates were successfully grown, four single colonies of each transformant were patched onto new agar plates. Individual colonies were selected using the tip of a round or flat toothpick and patched onto new agar plates with the appropriate antibiotics. The patch plates were grown in the 30 °C room for 2-3 days. When the patches had successfully grown, one patch from each transformant was inoculated into 500 µl of BG-11(N+) with Sp+Sm and Cm/Km. After the cultures had grown in the 30 °C room for 2-3 days, they were pipetted (1 µl) onto 16-chamber slides in preparation for imaging. Images were acquired using an inverted microscope with 100x objective lens and an exposure time of 0.1 seconds. Three different excitation channels were used: Differential Interference Contrast (DIC) for a bright-field image of the cells, Tetramethyl Rhodamine Iso-Thiocyanate (TRITC) for autofluorescence, and Green Fluorescent Protein (GFP) for detection of ZsG-CikA. The experimental settings for microscopy were established by Dr. Juliana Bordowitz.

Results

Successful transformation of all but 9 of the 2,426 library mutants was achieved with three screens. The combined localization screens resulted in 163 candidates that produced the mutant phenotype of mislocalized CikA. Microscope images showed that the mis-localized CikA phenotype could be present in all cells within a colony, or there could be a mixture of the mutant and wildtype phenotypes (Figure 3). Bioinformatic studies of the products of the 163 candidate genes indicate that some contain predicted transmembrane (TM) segments that may

Figure 2. Illustration of CikA localization screen. S. elongatus library mutants were transformed with the ZsG-CikA fluorescent construct from an E. coli donor. Transformants were selected for by antibiotic resistance to Sp+Sm (plasmid) and Cm/Km (insertional mutant), and imaged directly from the 96-well plate or from inoculated patches. Imaging from the liquid and solid media resulted in some overlap, but ensured complete coverage of the mutant library. (Image courtesy of J. Bordowitz)

anchor the protein essential for proper CikA localization at the cell pole. The protein’s C-terminus may localize in the cytoplasm or in the periplasmic space, a region between the cytoplasmic and outer membranes of gramnegative bacteria. Data were acquired from three transmembrane prediction servers, TMHMM, DAS, and TMpred (Figure 4), and a localization predictor, PSORTb (Table 1).6, 8, 9, 10

Discussion

For the initial localization screen, clones from the S. elongatus knock-out library were grown at a high light intensity of 322 µE in order to achieve a high throughput method. Clones that failed to transform initially were grown at a lower light intensity of 75-149 µE for the second and third passages of the screen. After using the latter conditions, 99.6% of the library was successfully transformed and subsequently imaged. The 9 clones that repeatedly failed to transform are believed to contain a mutation which could render the cell non-transformable. Images from the inverted microscope were taken using 3 channels; however, ﬁnal images contained only an overlay of the TRITC channel that showed a live cell’s

Table 1. Candidates from CikA localization screens. Candidates were noted as (P) for having mis-localized CikA like ∆PsR, or (M) for having a mixture of localized and mis-localized CikA. PSORTb predicted the C-terminus of the protein to be anchored to the cytoplasmic membrane, or in the periplasmic space.8 Predictions of cytoplasmic localization were of no interest to this experiment. Cyanobase predicted protein function based on known amino-acid sequence motifs. Period data was determined by J. Bordowitz after analyses of library screens done by C. Kay Holtman, unpublished. A circadian phenotype longer than that of wild-type (24 hours) is noted as a positive value while a shorter circadian phenotype is given a negative value.

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autoﬂuorescence, and the GFP channel showing the ZsG-CikA ﬂuorescent protein. Transformants from both agar and 96-well plates were imaged in order to maximize coverage. Inevitably, some overlap of candidates did occur. Among the 163 candidates, mutant and mixed phenotypes were observed. In the mutant phenotype, CikA was mis-localized because the protein needed for localization was lost. A mixed phenotype was noted in transformants that displayed localized and mis-localized CikA cells within a single colony. In the future, mixed-phenotypic candidates must be checked for single or double recombination events used for gene knock-out.7 An undesired single-recombination event would give the cell extra copies of genes that may have unanticipated effects on phenotype. Bioinformatic studies were conducted for each candidate using protein localization and transmembrane (TM) prediction servers. Protein localization predictions were given by PSORTb, while TM helix predictions were provided by TMHMM, Tmpred, and DAS servers (Figure 4).6, 8, 9, 10 A cytoplasmic prediction indicated that the protein was not anchored to the cell membrane, and thus of no immediate interest to this research project. Candidates that were predicted to localize to the cytoplasmic membrane or periplasmic proteins, which had predicted TM segment(s), were of greater interest. Such proteins would be anchored to the cell membrane, and able to localize CikA to the cell pole. Due to the great number of candidates, a select group of immediate interest has been prioritized for further studies. Priority was assigned to those with genes that could be readily analyzed. First, the mutant must show a clear phenotype of completely mislocalized CikA. Second, the knocked-out gene must be apart from or near the end of an operon, a cluster of genes under the control of a single promoter. If the mutated gene lies within an operon, it is unknown whether the mutated gene or a downstream gene is sq.ucsd.edu

RESEARCH Synechococcus elongatus serves to play a key role in future biofuels research.

Acknowledgements

We would like to thank Dr. Susan Golden for the opportunity to volunteer in her lab and Dr. Juliana Bordowitz for mentoring this research project.

References

Figure 3. Cells with localized and mislocalized CikA. Images show an overlay of autofluorescence from TRITC channel (red) and ZsG-CikA from GFP channel (green) that were acquired from an inverted fluorescent microscope using 100x objective lens. Bar is 5 μm. A) Wild-type with localized CikA. B) ∆PsR (negative control) with mis-localized CikA. C) UGS 8-D8 and D) UGS 10-A2 showing mislocalized CikA. E) UGS 10-E8 and F) UGS 11D3 showing a mixture of localized and mislocalized CikA.

responsible for the mutant phenotype, thus making further analysis a bit more complex. Finally, because CikA is a key input protein to the central oscillator, the degree of circadian period disturbance was also considered.

Conclusion

While the function of CikA in maintaining a circadian rhythm in S. elongatus has been documented, little is known about the partners that localize and anchor CikA to the poles of the cell. This study has revealed novel aspects of CikA localization by generating a list of potential partners believed to form an intricate protein complex with CikA. It is likely that multiple genes discovered from this screen may contribute to the localization of CikA; the precise number and mechanism is subject to further research. The knowledge gained from this study will help further understand the intricacies of circadian rhythms in living organisms. Cyanobacteria are a model organism for circadian research. The discovery of circadian rhythms within cyanobacteria has allowed for rapid advances in the ﬁeld, as the bacteria can be easily cultured and changes in metabolism and gene regulation can be measured. Knowledge of the internal mechanics of the circadian clock should also aid in the development of renewable algal biofuels. As a model organism that can anticipate the daily light:dark cycles required for photosynthetic activity and growth, sq.ucsd.edu

1. Dong, G., Golden, S. S. (2008). How a cyanobacterium tells time. Current Opinion in Microbiology, 11, 541–546. 2. Holtman, C.K., Chen, Y., Sandoval, P., Gonzales, A., Nalty, M.S., Thomas, T.L., Youderian, P., Golden, S.S. (2005). High-Throughput Functional Analysis of the Synechococcus elongatus PCC 7942 Genome. DNA Research, 12(2), 103-15. 3. Hughes, J. (2012). Phytochrome threedimensional structures and functions. Biochemical Society Transactions, 38, 710-716. 4. Mutsuda, M. (2003). Biochemical Properties of CikA, an Unusual Phytochrome-like Histidine Protein Kinase That Resets the Circadian Clock in Synechococcus elongatus PCC 7942. Journal of Biological Chemistry, 278(21), 19102-19110. 5. Zhang, X., Dong, G., Golden, S. S. (2006). The pseudo-receiver domain of CikA regulates the cyanobacterial circadian input pathway. Molecular Microbiology, 60(3), 658–668. 6. Cserzo, M., Wallin, E., Simon, I., VonHeijne, G., Elofsson, A. (1997). Prediction of transmembrane alpha-helices in procariotic membrane proteins: The Dense Alignment Surface method. Protein Engineering Design and Selection, 10(6), 673-676. 7. Finn, R.D., Mistry, J., Tate, J., Coggill, P., Heger, A., Pollington, J.E., Gavin, O.L., Gunesekaran, P., Ceric, G., Forslund, K., Holm, L., Sonnhammer, E.L., Eddy, S.R., Bateman, A. (2010). The Pfam protein families database. Nucleic Acids Research. 38(suppl 1), 211-222. 8. Yu, N.Y., Wagner, J.R., Laird, M.R., Melli, G., Rey, S., Lo, R., Dao, P., Sahinalp, S.C., Ester, M., Foster, L.J., Brinkman, F.S.L. (2010). PSORTb 3.0: Improved protein subcellular localization prediction with reﬁned localization subcategories and predictive capabilities for all prokaryotes. Bioinformatics, 26(13), 1608-1615. 9. Krogh, A., Larsson, B., Von Heijne, G., Sonn-

hammer, E. L. L. (2001). Predicting transmembrane protein topology with a hidden Markov model: Application to complete genomes. Journal of Molecular Biology, 305(3), 567-580. 10. Hofmann, K., Stoffel, W. (1993). TMbase - A database of membrane spanning proteins segments. Biological Chemistry Hoppe-Seyler, 374, 166.

About the Authors

Michelle Hoang is a Human Biology major from Earl Warren College. She will graduate in 2012. Stephen Leung is a Human Biology

and Psychology double major from Revelle College. He will graduate in 2012.

Figure 4. Example of bioinformatic graphs from transmembrane (TM) prediction servers. Amino acid position in the protein complex is designated on the x-axis. Probability of a TM segment is shown on the y-axis. A) TMHMM.9 B) TMpred.10 C) DAS.6 VOL 9 • SALTMAN QUARTERLY

SENI0R HONORS THESES

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www.sq.ucsd.edu

SENI0R HONORS THESES

SENIOR HonorS theses The Senior Honors Theses section is meant to recognize the achievements of the accomplished undergraduate researchers in the graduating class of 2012.

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SENI0R HONORS THESES

Senior HonorS Theses The Division of Biological Sciences Senior Honors Theses Program (BISP 196) is open to undergraduate biology majors who have an overall, and major, GPA of 3.7 or higher, have senior standing, and commit to three consecutive quarters of research during their senior year. The goals of the program are to increase one-one interaction between students and faculty to encourage more biology majors to pursue independent research. Each student in the program has a faculty mentor who provides guidance to the student during the year. In spring quarter each year, students in the honors thesis program participate in a poster session that showcases their research, and gives them the opportunity to discuss their research with faculty and their fellow students. Below are abstracts of all the outstanding research projects conducted by undergraduates in the program during the 2011-2012 academic year.

Investigating the cytokine activity of the Drosophila Toll Pathway

Allen Ninh, Warren College, Biochemistry and Cell Biology major, Psychology minor, Class of 2012 PI: Steven A. Wasserman Ph.D., Division of Biological Sciences, Section of Cell & Developmental Biology Comparisons between Drosophila species based on genome-wide transcription profiling have identified a core set of Toll-induced genes of unknown function that potentially play a role in the Drosophila innate immune response. We are currently developing assays to elucidate the function of these immune effectors and, for those that are likely to act as cytokines, identify their receptors. Ectopic expression of one candidate cytokine is associated with the proliferation of lamellocytes and melanotic tumor formation in larvae and adult flies. Bioinformatics studies suggest that this immune effector may act through a G protein-coupled receptor (GPCR) pathway. In order to study this, I have generated a stable cell line expressing a genetically encoded calcium indicator, GCaMP. Using a synthetic peptide and assaying for calcium influx in GCaMP expressing cells, we hope to identify the mechanism by which this immune effector acts. The results of these studies hold promise for providing a better understanding of these novel immune effectors and of their roles in the innate immune response.

Effects of Immunosuppresants Cyclosporin A and FK506 on Central Axon Outgrowth in vitro

Ari D. Kappel, Sixth College, Physiology and Neuroscience major, Cognitive Science minor, Class of 2012 PI: Mark H. Tuszynski, MD, Ph.D., Department of Neurosciences

Spinal cord injury (SCI) and disorders disrupt movement, sensation, and function of the central nervous system leading to lifelong debilitations and often paralysis. Unlike axons in the peripheral nervous system (PNS), injured axons in the adult mammalian central nervous system (CNS) do not regenerate after injury. Novel research in our lab has shown that grafts of embryonic nervous tissue can induce functional recovery of severed axons in the CNS. However, grafts of embryonic tissue require immunosuppression, and any future application of embryonic nervous tissue grafts in a clinical setting will require immunosuppression. In the past, it has been shown that conventional immunosuppressants such as cyclosporin (CsA), and prograf (FK506) inhibit axonal regeneration in the PNS (Tessier-Lavigne et al, 2003). The current research investigates the effects of CsA and FK506 on CNS axons in vitro. Time course information, and concentration dependence data are investigated in an isolated setting in order to elucidate the molecular and cellular effects of common immunosuppressants, CsA and FK506, on CNS axons.

Protective effects of spinach aldolase on muscle adenylate kinase and Phosphofructokinase-1 activities

Brian Jihoon Park, Muir College, Human Biology major, Class of 2012 PI: Dr. Percy Russell, Ph.D., Division of Biological Sciences

From previous studies, a hypothesis was developed that ascorbic acid (AA) inhibits muscle glycolytic enzymes during periods of rest to facilitate the storage of glucose as glycogen. Phosphofructokinase-1 (PFK-1) is considered the enzyme that controls the rate of glycolysis and along with lactate dehydrogenase (LDH) and adenylate kinase (AK), PFK-1 is inhibited by AA. During contraction, AA does not inhibit glycolysis due to the formation of a complex of glycolytic enzymes with contractile muscle proteins that protects them. Muscle aldolase prevents AA inhibitions and its protective properties are considered a microcosm of the complex formed with contractile muscle proteins. It was observed that spinach aldolase appeared to protect muscle PFK-1 activity from AA inhibition. The current study focuses on this ability of a plant enzyme, spinach aldolase, to interact and protect animal enzymes. Speciﬁcally the studies showed that spinach aldolase protected muscle AK activity loss due to dilution and protected muscle PFK-1 from inhibition by each AA and ascorbyl dipalmitate, an AA derivative shown to be more inhibitory than AA alone.

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SENI0R HONORS THESES Role of Tissue-nonspeciﬁc Alkaline Phosphatase and Fetuin in Biomineralization Bryan Lam, Revelle College, General Biology major, Urban Studies and Planning minor, Class of 2012 PI: Paul A. Price, Ph.D., Division of Biological Sciences

Our goal is to understand the mechanisms by which proteins control the the normal mineralization of bones and teeth and prevent the abnormal mineralization of soft tissues. We studied two proteins which have been implicated in the regulation of biomineralization: tissue-nonspecific alkaline phosphatase (TNAP) and fetuin. Fetuin is a 48kDa glycoprotein that is synthesized in the mammalian liver and found in high concentrations in serum and bone and is an important inhibitor of apatite growth in-vitro. TNAP has been identified in previous studies as a component of serum calcification activity and its effect in increasing the rate of mineral formation is observed only exclusively in the presence of fetuin. Apatite crystal formation was investigated using an in-vitro assay that closely resembles the calcium, phosphate and pH of human serum. We have demonstrated that fetuin is a substrate for alkaline phosphatase activity and that de-phosphorylation of fetuin greatly reduces its mineral inhibition activity. The high concentration of alkaline phosphatase and fetuin in bones suggest that bone fetuins may have a more dephosphorylated state. The reduced mineral inhibition activity of fetuin within bone may allow for normal mineralization to occur. Our findings also suggest that the phosphorylation state of serum fetuin may play a role in abnormal soft tissue mineralization such as the formation atherosclerotic plaques.

The role of the homeodomain protein Six6 in pituitary gonadotrope gene expression Chiara Maruggi, Revelle College, General Biology major, Italian Literature minor, Class of 2012 PI: Pamela Mellon, Ph.D., Department of Reproductive Medicine,

The hypothalamus orchestrates the release of FSH and LH from the pituitary through its pulsatile release of GnRH. FSH and LH in turn act on the gonads to stimulate hormonal secretions and gametogenesis. This research aims at better understanding regulation of gonadotrope gene expression through studying the mechanism by which Six6 represses the expression of LHβ, FSHβ, and GnRH receptor (GnRH-R). Six6, a member of the SIX/Sine oculis family of homeobox genes, stimulates progenitor cell development in the growing pituitary, eye, and brain. In mice, absence of Six6 causes a hypo-pituitary phenotype and a striking decrease in fertility. Recent work has shown repression of LHβ, FSHβ, and GnRH-R by Six6 and a relief of repression of the genes when transfecting with Six6 carrying a mutation in the eh1 domain. The region of repression by Six6 is in the 800 to 600 region for GnRH-R, in the -300 to -87 region for LHβ and inside of -95 in FSHβ. Two non-exclusive hypotheses will be explored further: displacement of activators from binding sites and recruitment of TLE co-repressors. The murine TLE (Grg, Groucho-related gene) proteins are co-repressors regulating development. Research has shown that both long-form (Grg4) and short form (Grg5) proteins regulate the GnRH gene by co-localizing with through Oct1 and Msx1 in vivo. Elaborating on these ﬁndings, this project also aims at identifying the possible role of TLE/Grg proteins in the repression of LHβ, FSHβ or GnRH-R. Such ﬁndings would help increase our knowledge of the mechanisms regulating GnRH release—mechanisms that play a key role in physiology and pathophysiology dealing with development and reproduction.

The Unique Shuttling Pathway of the Peroxisomal PTS2 Receptor, Pex7, in Pichia pastoris Danielle Hagstrom, Muir College, Molecular Biology major, Class of 2012 PI: Suresh Subramani, Ph.D., Division of Biological Sciences Peroxisomal matrix protein import relies on two pathways that use one of two peroxisomal targeting signals (PTSs), known as PTS1 and PTS2, present on cargo proteins. The majority of import occurs through the PTS1 pathway, dependent on the receptor Pex5. The PTS2 pathway is dependent on the receptor Pex7 and its co-receptor Pex20. Pex5 and Pex20 bind their respective cargo in the cytosol and shuttle them into the peroxisome matrix to be released. After cargo release, Pex5 and Pex20 are subject to mono-ubiquitination which allows for receptor recycling – a process dependent on the ubiquitin-conjugating enzyme, Pex4, and the AAA ATPases, Pex1 and Pex6. When receptor recycling is blocked, Pex5 and Pex20 are poly-ubiquitinated and degraded by the proteasome, in a process called the RADAR (Receptor Accumulation and Degradation in the Absence of Recycling) pathway. However, we found that the shuttling pathway of Pex7 differs from that of Pex5 and Pex20 as Pex7 is present at low levels in wild-type cells but stabilized in pex mutants affecting normal peroxisomal matrix protein import, suggesting that Pex7 is constitutively degraded during its normal import cycle. Speciﬁcally, Pex7 is stabilized in the receptor recycling mutants, Δpex4 and Δpex6, due to its inability to enter into peroxisomes, as seen by fractionation and protease protection assays. However, in the mutants affecting peroxisomal membrane proteins containing a RING domain, such as Δpex2, Pex7 can enter peroxisomes but presumably cannot be exported, thus causing accumulation of Pex7. Additionally, the import and export of Pex7 may have a previously unknown dependence on Pex5 and Pex20, which may be mediated by the special cargo, Pex8, which contains both a PTS1 and PTS2. The shuttling pathway of the PTS2 receptor, Pex7, demonstrates a sharp divergence from the previously studied shuttling pathways of Pex5 and Pex20 and could represent a mechanism to regulate Pex7 levels when the PTS2 pathway is not needed.

Role of Calcium Sensors in Stress Responses of Plants

Desiree Nguyen, Thurgood Marshall College, Biochemistry and Cell Biology major, Psychology major, Class of 2012 PI: Julian Schroeder, Ph.D., Division of Biological Sciences Stomata, present in the aerial epidermis of land plants, provide gateways for regulating carbon dioxide (CO2) and water exchange between plants and the atmosphere. The stress induced phytohormone abscisic acid (ABA) reduces transpirational water loss crucial for the ﬁtness of plants by inducing stomatal closure . Cytosolic Ca2+ has been reported to play a major role in ABA-induced stomatal closure. The objective is to elucidate the roles of members of two different protein families that represent calcium sensors in stress responses of the model plant

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SENI0R HONORS THESES Arabidopsis thaliana - Calcium Dependent Protein Kinases (CPKs) and Calmodulin-like (Cml) proteins. cpk3/6 and cpk 4/11 double mutant plants show impaired ABA- and Ca2+-induced stomatal closing and Cml9 knock-out plants (cml9-1 and cml9-2) are reported to be drought sensitive and show an ABA-dependent seedling growth phenotype. Underlying molecular mechanisms leading to these altered responses are still poorly understood. To improve the understanding of these proteins and their mechanisms, two different quadruple mutant plant lines, cpk3/cpk4/cpk6/cpk11 and cpk5/cpk6/cpk11/cpk23, will be established by crossing parent plant lines, and subsequent screening using PCR based genotyping. Gene expression knock-out will be conﬁrmed by RT-PCR. For both, the quadruple CPK and the Cml9 knock-out lines bioassays determining stomatal movements in response to several stimuli will be carried out improving the understanding of the role of these proteins within plants’ abiotic stress tolerance to environmental challenges, such as droughts. Consequently, strategies can be developed and engineered to promote strong resistance against environmental stresses in plants, resulting in increased crop yields.

Exploring the Regulation of yidC2 in Bacillus subtilis George Chen, Revelle College, Biochemistry and Cell Biology major, Class of 2012 PI: Kit Pogliano, Ph.D., Division of Biological Sciences The YidC/Oxa1/Alb3 family of proteins found in bacteria, mitochondria, and chloroplasts respectively, perform the essential function of mediating the folding and insertion of transmembrane proteins into their proper locations in the membrane. In Gram-negative bacteria, only one copy of YidC is present; in Gram-positives, there are two, SpoIIIJ/YidC1 and YidC2. Previous research in the Gram-positive species, Streptococcus mutans and Bacillus subtilis has shown that although spoIIIJ/yidC1 and yidC2 share many overlapping functions, they also differ in several ways. Our goal is to determine the conditions that cause B. subtilis to express yidC2 in preference to the constitutively active spoIIIJ. We performed a transposon mutagenesis to isolate mutants that upregulate the yidC2 operon. This screen led to the discovery of several mutants with insertions in the qox operon, that codes for the menaquinol oxidase terminal electron acceptor, cytochrome aa3-600, as well as abrB, a transition state regulatory gene. In addition, Phenotype Microarrays were performed to identify differences in metabolism and chemical sensitivities among ∆spoIIIJ, ∆yidC2 and wild type strains. We observe alterations in sensitivity to a number of compounds, with several affecting membrane integrity and redox balance. These results suggest that changes affecting redox and oxidative metabolism may lead to an upregulation of yidC2.

The RNA-Binding Protein Musashi Regulates Epithelial-Mesenchymal Transition and Invasion in Head and Neck Squamous Cell Carcinoma Elham Rahimy, Revelle College, Biochemistry and Cell Biology major, Class of 2013 PI: Martin Haas, Ph.D., Division of Biological Sciences The cancer stem cell hypothesis posits that within a tumor exists a distinct subpopulation of cells responsible for tumor initiation, progression, and maintenance. These cancer stem cells (CSCs) tend to be resistant to conventional chemotherapy and, similar to normal stem cells, possess the ability to self-renew and differentiate into the heterogeneous lineages comprising a tumor, thereby causing tumor recurrence. Emerging evidence in support of the cancer stem cell hypothesis suggests that genes responsible for pluripotency and self-renewal in embryonic stem cells may also be essential for the maintenance of cancer stem cells. The Musashi family is a group of conserved RNA-binding proteins previously shown to up-regulate expression of Notch through translational repression of mammalian Numb (m-Numb) by interacting with the 3′-untranslated region (UTR) of Numb mRNA. The Notch-activated signaling cascade is a known regulator of self-renewal, differentiation, cell proliferation, apoptosis, invasion and migration in stem cells, and thus a potential therapeutic target for CSCs. Concurrent knockdown of two Musashi isoforms in neural stem cells (NSCs) results in inhibition of tumorsphere formation in non-adherent and non-differentiating conditions. Given the importance of Musashi as a stem cell marker and the implications of the cancer stem cell hypothesis, we sought to determine the role of Musashi in head and neck squamous cell carcinoma (HNSCC) invasion, as well as to determine the underlying mechanism. We ﬁrst show that Musashi shows markedly higher expression in a putative HNSCC CSC culture as compared to four other HNSCC cell lines and a positive control, the breast cancer cell line MCF-7. Upon transient overexpression of Musashi in two of the HNSCC cell lines, invasion was signiﬁcantly up-regulated. As induction of epithelial-mesenchymal transition (EMT) is considered a critical process for the acquisition of an invasive phenotype, we then decided to study the effects of Musashi on EMT. Musashi overexpression induced the expression of several EMT markers, and down-regulated expression of the epithelial marker E-cadherin, a surface protein critical for cell-cell adhesion. We are currently investigating the role of long intergenic non-coding RNA (lincRNA) in mediating Musashi-induced HNSCC invasion.

Three New Species of Tripleﬁn Blennies with Notes on the Tripterygiidae of the Tropical Eastern Paciﬁc

Elizabeth C. Miller, Thurgood Marshall College, Evolution, Behavior, and Ecology major, Marine Science minor, Class of 2012. PI: Philip Hastings, Ph.D., Marine Biology Research Division, SIO. Ron Burton, Ph.D., Marine Biology Research Division, SIO

The triplefin blennies are cryptic, bottom-dwelling fishes with three dorsal fins that are found worldwide. Three new species of the genus Enneanectes (Teleostei; Tripterygiidae) are described from the Baja California and the Pacific coast of Mexico. These species were identified as distinct from other members of the genus in 1959 using morphological data; however, the species descriptions were never published. Additional morphological analysis including specimens from the Marine Vertebrate Collection (SIO) confirms that two of the three species are easily distinguishable from congeners. Species A is found from the southeastern Gulf of California to southern Mexico, and is distinguishable by brown reticulations outlining scales and 15 pectoral fin rays (with lower 7 unbranched). Species C (common name: chacala blenny) is more elongate and has higher meristic counts in all characters measured than other members of Enneanectes. Its range has been extended through Southern Mexico. Species B was identified as endemic to the

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SENI0R HONORS THESES Revillagigedos Islands, though its classification as a distinct species is pending molecular analysis. The phylogenetic relationships of the Tripterygiidae of the Tropical Eastern Pacific are poorly resolved. A continuation of this project will attempt to construct a phylogeny of the Tripterygiidae of the Tropical Eastern Pacific using sequence data from the following genes: CO1, Rhodopsin, Cytochrome b, 16S, 12S and 18S. Taxon sampling will cover twelve species from four genera, including those formally described by this project.

Novel Role of Mannose Binding Lectin in Age Related Neuroinflammation and Neurocognitive Consequences in HIV Infected Brain Dong Tran, Eleanor Roosevelt College, Biochemistry and Cell Biology major, Philosophy major, Class of 2012 PI: Kumud K. Singh, Ph.D., Department of Pediatrics The advent of HAART has led to a larger number of individuals with HIV surviving into old age. While aging alone leads to a weakened immune system, immune complex deposition, and neuroinflammation in the brain, previous studies have found that HIV infection worsens these effects. Among the synergistic effects is an increased deposition of beta amyloid (BA), a protein associated with Alzheimer’s disease. Complement activation is a crucial part of innate immunity that recognizes and initiates an immune response to pathogens via three different pathways. The lectin pathway, mediated by mannose binding lectin (MBL), is one such pathway. MBL can bind to mannose residues on HIV-1 GP120 envelope protein, leading to complement activation. Previous work has shown that there is an increased MBL expression in individuals with HIV encephalitis vs. non-HIVE and that there is an overall activation of lectin complement pathway in HIVE. An age-dependent association of neuroinflammation and neurocognitive consequences with MBL mediated complement activation has not been shown. Here, we examined the expression and colocalization, via Immunofluorescence, of MBL, GP120, BA, and MBL associated serine protease-2 (MASP-2), an enzyme that is a marker of lectin pathway mediated complement activation, in post-mortem brain tissue derived from HIV+ individuals with/without HIVE and HIV- individuals. Our preliminary results show that there was an increase in MBL, BA, and MASP-2 expression and colocalization with increasing age in HIVE vs. non-HIVE/HIV+ and in non-HIVE/HIV+ vs. HIV- cases. In summary, an age dependent increase in colocalized MBL, BA and GP120 in HIVE as compared to non-HIVE/HIV+ was observed. Thus, there is evidence that MBL and MBL mediated complement activation plays a part in HIV disease progression, with implications that MBL functions in immune complex deposition with BA and HIV viral protein. Future work will focus on further investigation of MBL pathway and related immune response in age dependent neuroinflammation and neurodegeneration in HIV-1 infected individuals.

Characterizing Divergent Bacteriophage-Encoded Tubulin Homolog “PhuZ” Hannah Wang, Muir College, General Biology major, Literatures of the World minor, Class of 2012 PI: Joe Pogliano, Ph.D., Division of Biological Sciences. We have identified and characterized a protein encoded by a bacteriophage that shares high sequence homology with divergent prokaryotic tubulins FtsZ and TubZ. Named PhuZ, short for Phage TubZ, this divergent tubulin is synthesized by 201ϕ2-1, a large dsDNA myoviridae phage that infects bacterial host Pseudomonas chlororaphis. While the crystal structure of PhuZ, determined by the Agard Lab at UCSF, greatly resembles that of FtsZ, its C-terminal hook-like tail is unique. From structurebased interaction predictions, we hypothesize that this tail plays a crucial role in filament assembly. Through fluorescence microscopy and growth curve experiments comparing the wild type protein with proteins mutated at amino acids expected abolish tail function, we have begun to characterize this filament-forming tubulin-like protein encoded in the genome of a bacteriophage. The results suggest that PhuZ, like TubZ, polymerizes into filaments and exhibits treadmilling in vivo. However, it is singular in that its C-terminal hook-like tail plays a crucial role in its assembly. Mutations at this site increase the critical concentration for assembly, but assembled polymers show the same dynamics as wild type. The study of PhuZ has uncovered new possibilities in our understanding of tubulin polymer assembly and in our conception of how large phage successfully replicate and lyse their hosts.

Identification and Characterization of a Regulatory Mutation that Causes Hypervariable Phenotypes in Drosophila Embryos Jasmine Ong Manubay, Muir College, Human Biology Major, Studio Arts minor, Class of 2012 PI: Steven Wasserman, Ph.D., Division of Biological Sciences The overall goal of my project is to study the molecular basis of variable gene expression, with a gene in the Toll signaling pathway serving as the experimental system. The Toll pathway is highly conserved between both mammals and flies. In Drosophila melanogaster, one of the functions of this pathway is to specify ventral fate in embryos. When this pathway is interrupted, embryos display a dorsalized phenotype. The severity of such dorsalization can range from severe to mild, depending on the degree to which Toll signaling is decreased. One gene in this pathway is Tube, which encodes an adaptor protein. Most mutations in the Tube gene lead to a set level of dorsalization, whether it be strong or weak. I am studying the tub8 allele, which causes single female flies to produce embryos that range across the entire spectrum of dorsal phenotypes (from D0, the strongest degree of dorsalization, to D3, the mildest phenotype). Previous research show that tub8 mutants contain no sequence change in the mature protein. However, quantitative PCR results reveal a decreased amount of transcript, suggesting that a regulatory mutation might be the cause of decreased signaling. My experimental focus is to map and identify this regulatory mutation using site-specific recombination in combination with sequencing. To date, I have mapped the regulatory mutation to a 16 kilobase region containing the Tube gene. Using sequencing, I will identify the regulatory mutation and subsequently determine the molecular mechanisms of variable transcriptional repression of the Tube gene. These results may help the understanding of transcriptional regulation and stochastic processes in the cell, as well as the molecular mechanisms of variable phenotypes that occur in numerous organisms including humans. www.sq.ucsd.edu

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SENI0R HONORS THESES In-vivo expression analysis of potential virulence genes of Leptospira interrogans in a hamster model of infection Jason Tanseco, Revelle College, Biochemistry and Cell Biology major, Class of 2012 PI: Joseph Vinetz, M.D., Department of Medicine, Molecular Pathology

Leptospirosis is a widespread zoonotic disease caused by virulent spirochaetes of the genus Leptospira. Despite over 1 million severe cases of Leptospirosis occurring annually and case mortality rates approaching 20%, much remains unknown with regards to its molecular mechanisms of pathogenesis. Previous experiments aimed at elucidating virulence factors using comparative genomics between saprophytic and virulent Leptospira, transposon mediated mutagenesis, and microarray studies under various environmental conditions have been largely unsuccessful. To date, only 5 virulence factors have been identiﬁed for pathogenic leptospires: a lipopolysaccharide, heme oxygenase, ﬂagella, InvA, and the OmpA-like Loa22. In an effort to characterize additional virulence factors, an attenuation experiment was conducted using a highly pathogenic (LD50 < 100 bacteria) strain of L. interrogans serovar Lai that was passed bi-weekly until it became avirulent (LD50 >108 bacteria). Both genomes were then sequenced using Solexa nex-gen sequencing. This revealed that the attenuated organism had accumulated 35 single nucleotide polymorphisms (SNPs) resulting in non-synonymous, protein coding amino acid substitutions. Next, an experiment was conducted on the original virulent strain to determine if the RNA products of these genes were upregulated in a host organism using relative changes in fold expression with reverse transcriptase-PCR, resulting in robust upregulation of many of the candidate genes.

Comparison between the effects of spinach aldolase and rabbit muscle aldolase on LDH Jin Kang, Eleanor Roosevelt College, Biochemistry and Cell Biology Major, Class of 2012 PI: Percy Russell, M.D., School of Medicine

It was shown that the enzyme, aldolase, from rabbit muscle protected muscle phosphofructokinase-1, muscle adenylate (AK), and lactate dehydrogenase (LDH) from inhibition by ascorbate (AA) and some AA-fatty acid derivatives. Preliminary studies showed that a plant enzyme, spinach aldolase, had similar properties. This study extends the protective activities of spinach aldolase with respect to muscle LDH and introduces an interesting topic of the relationships between plant and animal proteins in terms of evolution. Rabbit and spinach belong to different Kingdoms and it would not be expected that their proteins would interact. Experiments comparing protective effects of rabbit muscle aldolase and spinach aldolase on inhibitions of LDH by AA and some AA-fatty acid derivatives were conducted. In addition to the inhibitor AA, the following AA-fatty acid derivatives were also studied: ascorbyl phosphate (AAPO4), ascorbyl stearate (AAS), and ascorbyl dipalmitate (AADP). It has been discovered that they have very similar protective behavior for LDH against inhibitions by dilution, AA, AAP, AADP, and AAS; however, spinach aldolase seems to protect and recover the activity of LDH better than rabbit muscle aldolase does. It is believed that this and related basic science research has some relation to cancer and diabetes studies.

The role of secondary metabolites in the interspecies interactions of Serratia marcescens and Bacillus subtilis

Michael Dean Sharp, Revelle College, Molecular Biology major, Class of 2012 PI: Kit Pogliano, Ph.D., Division of Biological Sciences We investigated the interspecies interaction between two soil bacteria, Bacillus subtilis, which is used as a probiotic, and Serratia marcescens, an opportunistic pathogen. We found that B. subtilis and S. marcescens form a co-colony when grown alongside one another. However when either one of the species is placed at a higher concentration, relative to the other, it is able to inhibit growth of the other species. Therefore the outcome of this interspecies interaction varies under different conditions. We tested transcription factor mutants of B. subtilis and found a biofilm development gene, sinR, and two environmental stress response genes, ylaC and sigB, to be necessary to inhibit growth of S. marcescens. We also tested secondary metabolite mutants of B. subtilis and discovered two genes to be necessary for B. subtilis to inhibit growth of S. marcescens; sfp, which is necessary for synthesis of several secondary metabolites, and pksX, responsible for a polyketide synthase which synthesizes bacillaene. In order to isolate secondary metabolites from each species we performed solid-phase extraction of bacterial cultures grown both independently and in interaction. Matrix assisted laser desorption ionization – time of flight (MALDI-TOF) mass spectrometry was performed to identify the masses in all the fractions obtained from solid phase extraction, and correlate specific masses to the fractions that cause growth inhibition. Further purification by High Purification Liquid Chromatography (HPLC) of those fractions indicate that S. marcescens produces at least two metabolites capable of inhibiting B. subtilis growth. Specifically, we discovered purified prodigiosin, a secondary metabolite from S. marcescens, to strongly inhibit growth of B. subtilis. Secondary metabolites are known to contribute to bacterial survival and defenses in interspecies interactions, and the work presented here further elucidate the variety of impacts that secondary metabolites can have in nature

Olfactory imprinting in Drosophila Lea Currier, Thurgood Marshall College, Physiology and Neuroscience major, Class of 2012 PI: Jing Wang Ph.D., Division of Biological Sciences, Section of Neurobiology During a sensitive period, usually early in the life of many animal species, sensory experiences play an important role in shaping foraging patterns and habitat selection. Sensory cues signaling for safety and for food are quickly learned by young animals and encoded in their memories through modification of neurons in the sensory neural circuit. Imprinting of sensory cues is an important survival mechanism and a rudimentary form of learning. The imprinting of sensory cues can guide and shape an animal’s preferences and behavior. However, it is not well understood how early sensory experiences can contribute to the neural plasticity that influences behavior and thus has been the focus of the present study. Our results demonstrate that olfactory preference in adult Drosophila is markedly influenced by odor experiences during critical period—the first two days following eclosion. Exposure to apple cider vinegar during the critical period dramatically increases the flies’ preference for that odor in the later stages of the animal’s life. We found that conditioned responses are abolished in Or83 mutant flies, suggesting that olfactory receptor neurons (ORNs) function is required for olfactory conditioning.

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SENI0R HONORS THESES We aim to isolate the neural substrate that mediates the acquisition and retrieval phase of conditioned response. We found that blocking Or42b ORN output during acquisition abolishes olfactory conditioning suggesting that Or42b ORN output is required in the acquisition phase. In addition, ﬂies do not exhibit increased behavioral response towards apple cider vinegar in the absence of Or42b ORN output during testing. This indicates that Or42b ORN output is required for retrieval. In conclusion my work provides a causal link between sensory neural plasticity and behavioral modiﬁcation.

Intron retention and alternative AUG site usage characterize the unique regulation of a glycolytic transcription factor in response to glucose availability in S. cerevisiae Mary Fok, Muir College, Human Biology major, Class of 2012 PI: Tracy Johnson, Ph.D., Division of Biological Sciences, Section of Molecular Biology

In higher eukaryotes, regulated alternative splicing is a critical means by which cells achieve genomic reprogramming in response to environmental changes. In the yeast Saccharomyces cerevisiae, genome-wide microarray analyses indicate that splicing outcomes are affected under different stresses, but our understanding of how splicing is regulated in response to a dynamic environment is still limited to only a handful of genes. Notably, each of these genes contains an intron with a non-consensus splice site. We identiﬁed GCR1 as a novel candidate for regulated splicing in yeast since the GCR1 intron contains a non-consensus 5’ splice site and expression of Gcr1 is sensitive to glucose availability. Here we examine how expression of Gcr1 is regulated in response to glucose at the level of splicing, RNA decay, and translation. Surprisingly, we observe that the GCR1 intron is preferentially retained in the presence of glucose and that an alternative start codon in the GCR1 intron permits the generation of a 94kDa protein. The levels of this protein decrease rapidly upon glucose depletion, as do the levels of the GCR1 pre-mRNA, while the levels of the spliced GCR1 mRNA remain unchanged. Deleting the cytoplasmic decay factor, Xrn1, also affects the ratio of spliced to unspliced GCR1 suggesting that the different GCR1 isoforms are subject to multiple levels of regulation. This work provides the ﬁrst example of alternative AUG site usage that depends on regulated intron retention in yeast.

Comparison of the activities of the seven human TUTases

Jong Hyun Park, Eleanor Roosevelt College, Human Biology major, Class of 2012 PI: Jens Lykke-Andersen, Ph.D., Division of Biological Sciences Terminal uridylyl transferases (TUTases) are template-independent polymerases capable of catalyzing the transfer of single or multiple UMP residues to RNAs in eukaryotes. Previous studies have shown that these enzymes play a role in messenger RNA and small RNA turnover. The in vitro activities of a few of the seven human TUTases (TUTase 1 - TUTase 7) have already been characterized, but done so individually. Here we compared the activities of ﬁve of the seven human TUTases. Our data indicate that, in vitro, TUTase 4 and 7 prefer to add UMP residues to an RNA template, while TUTase 2, 3 and 5 prefer to add AMP residues. Furthermore, we show that TUTase 7 is unique in its ability to not only add short tails to an RNA template, but also very long tails over 100 nucleotides long.

Moderation of Heart Failure and Depressive Mood by Immunosenescence

Tiefu Cheng, Eleanor Roosevelt College, Physiology and Neuroscience major, Psychology major, Class of 2012 PI: Suzi Hong, Ph.D., Department of Psychiatry Cardiac pathophysiology in the general public correlates with an elevated prevalence of depressive mood, the presence of which predicts morbidity and mortality in patients with conditions such as heart failure (HF). In existing literature on cardiovascular disease (CVD) and depression, the relationship between each illness to inflammation and immunoactivation has been well documented. However, inconsistent findings exist in describing inflammation via circulating cytokine levels as the bridge between current understandings of depression and CVD. The present study explores the possible relationship between depression, heart failure, and immunosenescence, which encompasses immune functional decline due to chronic immunoactivation. This decline is characterized by greater population percentage of terminally differentiated effector T lymphocytes, which detracts from the efficiency of the adaptive aspect of immunity. Adult participants diagnosed with Stage B HF as classified by the American College of Cardiology/American Heart Association were assessed for depressed mood via the Beck Depression Inventory (BDI-1A) and drawn for blood. Peripheral blood mononuclear cells were isolated and analyzed via flow cytometry after being stained with fluorochrome conjugated antibodies for surface markers (CD45RA, CCR7, CD3, CD8, CD57, CD28, CXCR1, CX3CR1). Crosssectional analysis will contribute evidence towards explaining the relationship between immunosenescence, Stage B HF, and depressive mood.

Innovative Synthesis of Molecular Rotors for Visualization of Amyloid Deposit and Measurements of Cytoplasmic Viscosity Thai Do, Muir College, General Biology major, Class of 2012 PI: Marianna Dakanali, Ph.D., Emmanuel Theodorakis, Ph.D. Department of Chemistry and Biochemistry,

Molecular rotors is a class of organic probes that fluoresce in an environment-dependent manner. In this study, we present two innovative uses of these probes. First, modifications to the molecular rotors motif were introduced to explore their capability to fluorescently label aggregated amyloid-Beta (Aβ) plaques in Alzheimer’s Disease. In ex vivo experimentation with human brain tissues from Alzheimer’s patient, these probes fluorescently labeled Aβ peptide in low micromolar concentration. Second, we present the synthesis and spectroscopic characterization of a ratiometric, self-calibrating hydrophobic dye, as non-mechanical viscosity sensor. Cell localization experiments of this dye suggest that it cross the cell membrane and potentially can be used for viscosity measurements in the cell. With this study, we envision the development of these probes as low cytotoxic and highly fluorescent visualization tools to study biological and physiological environment common in human diseases. www.sq.ucsd.edu

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SALTMAN DEDICATION

Remembering Paul Saltman written by

Lawrence Ku & Nishita Shah

his legacy as a biochemist and a teacher...

magine sitting in a lecture hall listening to the A respected Biochemistry professor and nutrition professor drone on and on about the pathways governing researcher, Dr. Paul Saltman was a big advocate of glycolysis and fatty acid oxidation. Your mind starts to educating the public on good choices when it came to drift, when all of a sudden, you are jolted awake by the nutrition. His layman’s guide to food, The California booming voice of a six-foot-ﬁve, towering professor Book of Nutrition, demystiﬁed and debunked the writing on the chalkboard. You almost forgot that this many diet fads in the 1970s and allowed the public to is Dr. Saltman’s lecture, and that this 8 a.m. class is no understand the science behind nutrition. As a biochemist, time to daydream. Pulling out your bag of M&M’s for Saltman was a big believer in maintaining a balance of breakfast, you begin to munch on the rainbow-colored foods for a healthy lifestyle. Nutrition was much more candies as you learn about what happens to each glucose than just checking laundry lists of calorie, sugar, and fat molecule as it enters your body. Wondering how much consumption; it was a series of choices each individual ATP you will get from this little bag of chocolate, you take needed to make for themselves. Food interacted with a look at the nutrition label on the back and are shocked one another, and thus blanket statements about XX at the amount of fat, calories, and sugar you just put into your mouth. And all just to satisfy that growling stomach and ever pesky sweet tooth of yours. But then Dr. Saltman says something that not only eases your worry of gaining a little weight, but also changes the way you view your eating habits: “There is no such thing as junk food; only good or bad choices.” He goes on to discuss how no one ever became overweight just by eating a few chips here and there, but rather became unhealthy through nutritional imbalances in their diets. Feeling relieved that an expert on the matter just declared your M&M’s to be safe for further consumption, you start to reevaluate A typical sight during his teaching years: Dr. Saltman hanging out with students the way you view food and nutrition. outside of class to discuss school and prospective future plans.

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SALTMAN DEDICATION again. Saltman, doing extensive trace mineral research at the time, worked with Walton and tried to adjust the vegetarian basketball player’s diet so that it accommodated more copper, zinc, and manganese. Bones would be unable to properly build without the necessary levels of manganese and copper, and they could not heal without enough zinc, even if the body had enough calcium. With Saltman’s trace mineral concoctions, Walton’s foot healed, and he was back shooting hoops on the court within two months. The key to treating the poor bone condition was nutritional balance, and as Saltman always stressed, it is the An amazing lecturer, Dr. Saltman not only motivated students to love and balance of nutrients – in Bill Walton’s case, understand biochemistry but also to think about their own life goals, and how micronutrients – that will eventually heal you. nutrition related to their everyday lives. Dr. Linda Strause, Professor of Nutrition surely could not apply to everyone. Saltman’s “no- at UC San Diego, worked alongside Saltman in the 1980s nonsense” approach was designed to teach you how on his trace mineral research. As a postdoctoral fellow to understand nutrition and apply this knowledge to in his lab, Strause had the opportunity to learn not only your food intake in times of health and disease. about the research Saltman conducted, but also his own Not only did Saltman have a huge impact on the philosophies regarding teaching and education. “He was a way society viewed nutrition, but he also changed the great mentor; he believed in both education and hard work, individuals he worked with, students and athletes alike. and that if you had both you would be phenomenal,” said Bill Walton, Basketball Hall of Fame inductee who had Strause when asked about Dr. Saltman inﬂuence on her life. led the Portland Trail Blazers to a Cinderella story NBA Encouraging her throughout her career, Saltman gave championship, had multiple, recurring foot injuries due Strause the conﬁdence and support she needed to excel to a bone condition that resembled osteoporosis. Due to as both a biochemist and a teacher. His real passion was these fragile bones, Walton was forced to take time off giving the gift of knowledge to others, and he believed playing for the San Diego Clippers in the early 1980s, and that by teaching and learning, you could attain true it seemed as if he would never be able to play basketball freedom. True to his philosophy on nutrition, Saltman similarly believed that there needed to be another balance in life, one between education and hard work. With both, it would be possible to accomplish anything, even a healthy body, so that one could live a productive life. This philosophy was applied across all of Saltman’s legacies, teaching and research alike. He believed heavily in the science behind nutrition because by educating the public about the nutrients they need, individuals could make their own decisions regarding the lifestyles they choose. Similarly, Saltman invested his efforts into education, teaching with a passion and vigor that allowed each of his students to gain the knowledge to make their own decisions in life. Saltman once declared, “One reason we teach is that it keeps us immortal.” And in this tradition, this journal keeps his spirit alive by teaching students year after year about science and communication. Dr. Saltman and Dr. Linda Strause check for supplies in the lab for a trace mineral study. sq.ucsd.edu

VOL 9 • SALTMAN QUARTERLY

Staff Editor-in-Chief Lawrence Ku

Senior Technical Editor for Content Esther Oh

Communications/Publicity Chair Jarod Corey

Managing Editor Hilary Lee

Junior Technical Editor for Content Annie Wang

Senior Research Editor Payal Desai

Technical Editor of Graphics Erica Couzens Sabrina Trinh

Communications/Publicity Committee Mallika Maileswaram Shruti Patel

Junior Research Editor Vicky Hwang Features Editor Sara Mokhtary Production Editors Shawn Shah Nishita Shah

Research Design Editor Sameeha Khalid Features Design Editor Kriti Gupta Review Board Managers Kit Wu Milli Desai

Faculty Advisory Board

Staff Writers: Anelah McGiness Iliana Nguyen Joseph Aleshaki Kevin Perez Preuss School Staff Writers: Lucy Luong Sharon Vongvanith

Staff Advisors

Cell & Developmental Biology Jim Wilhelm

Associate Dean for Education Gabriele Wienhausen

Ecology, Behavior, and Evolution Teri Markow Heather Henter

Media Specialist, Dean’s Office Katie Frehafer

Molecular Biology Eric Allen James Golden

Undergraduate Advisor, Student Affairs Hermila Torres

Neurobiology Jill Leutgeb

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Ecology Behavior and Evolution Aditi Abraham Alexandra Delaney Allene Chang Alpha Joy Inton Amirhossein Jaberi Andrew Tu Caitlin Riley Carole Wang Carolyn Kim Chelsea Abang Chris Law Desiree Nguyen Elizabeth Stringer Hilary Lee James Kang Jamie Lii Jessica Lau Johnson Jou Jonathon Chan Kevin O. Perez Lily Huang Mallika Malleswaran Mersedeh Mousacohen Polina Brodsky Shaham Kamkar Shuin Park Tiffany Tran Zachary Hodosevich Molecular Biology Adena Pepich Adrian N. Van Sickel Alex Muselman Brian J. Chang Brian Tran Cory Jameson

Review Board Dimple Patel Dominick Lau Elizabeth Chiang Govind Raghavan Jaimie Long Jeiran Javaherian Jonathan Day Julie Shen Jung Yeon Kim Karl A. Hujsak Kevin Cheung Mikiko Bazeley Neil Raina Pial Hossain Robert Betz Sammy Chang Sara Mokhtary Sharon Hsu Sonia Kim Sonya Subash

Cell & Development Biology Andrew Kim Andrew Lum Angela Shen Arlene Ngor Cris Molina Diana Dabbous Erica Couzens Francisca Ryani Gabriella R. Marvizi Goutam Krish Jui E. Chang Katherine Moran Kian Nassiri Kritika Reddy Laura Toy

Matthew Mayeda Michael Pimienta Namrata Vora Nitin Prabhakar Peik L. Sia Rosanne Luu Sher Khan Tu Nguyen Viktoriya Megrabova Yasaman Pirahanchi Neurobiology Aaron Kappe Alison Uyeda Anne Salib Ari Kappel Aya J. Sangen Chris Probert Diana Ponce-Morado Farzad Hasnat Gina Nalbandian Howard Nguyen Jacky Lin Jeff Sobieraj Joanna Coker Joseph Giessinger Julie A. Dang Jun Kyoung Karen N. Fernandes Laura Schellenberg Lily Huang Mehulkumar Dhanani Oly Khowash Randy Tran Rasaena Williams Seung Jin Lee Sora Chee Tricia Ngo

Top Row: Jarod Corey, Kit Wu, Nishita Shah, Payal Desai, Shawn Shah, Milli Desai, Erica Couzens, Kriti Gupta, Esther Wang Middle Row: Sameeha Khalid, Annie Wang, Sara Mokhtary, Hilary Lee, Iliana Nguyen, Dr. Gabriele Wienhausen, Katie Frehafer, Bottom Row: Anelah McGinness, Vicky Hwang, Lawrence Ku, Joseph Aleshaki, Kevin Perez, Mallika Maheswaran Not Pictured: Lucy Luong, Sharon Vongvanith, Shruti Patel, Sabrina Trinh

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Saltman Quarterly Division of Biological Sciences University of California, San Diego 9500 Gilman Drive La Jolla, CA, 92093-0376 (858) 534-5635